Electrochemical sensors

ABSTRACT

An electrode for use in a electrochemical sensor comprises carbon modified with a chemically sensitive redox-active compound, excluding an electrode based on carbon having derivatised thereron two redox-active species wherein at least one of said species is selected from anthraquinone, phenanthrenequinone and N,N′-diphenyl-p-phenylenediamine (DPPD). The invention further provides a pH sensor comprising:
         a working electrode comprising carbon modified with a chemically sensitive redox active material; and   a counter electrode,
 
wherein the ratio of the surface area of the working electrode to the surface area of the counter electrode is from 1:10 to 10:1. Also provided is a pH sensor comprising:
   a working electrode comprising carbon modified with a chemically sensitive redox active material, and   a counter electrode,
 
wherein the area of the working electrode is from 500 μm 2  to 0.1 m 2 . The uses of these electrodes and sensors are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/183,632, filed Jul. 15, 2013, which is a continuation of U.S. patentapplication Ser. No. 10/591,491, filed Sep. 1, 2006, abandoned, which isa national stage application under 35 USC 371 of PCT Application No.PCT/GB2005/000802, filed, Mar. 4, 2005, expired, the disclosures of bothof which are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to electrochemical sensors for use in variousenvironments, including non-downhole environments. For example, thesensors may be used for the determination of pH of substances in “dirty”environments, such as effluent and other waste streams the inventionalso relates to various materials, which may be used, for instance, inelectrodes forming part of electrochemical sensors.

BACKGROUND OF THE INVENTION

U.S. Pat. No. 5,223,117 relates to self-assembly microelectrodes used inelectrochemical sensors. The microelectrodes (which are the workingelectrode in the sensors) are modified with monolayer coverages ofreference and indicator molecules, with both chemically sensitive redoxmaterials and chemically insensitive redox materials being present onthe same electrode. It is necessary for the microelectrodes to besignificantly smaller than the counterelectrodes, for example thecounter electrode area must be at least 102 to 103 times the workingelectrode area. An advantage of such a small working electrode with aninternal reference is that the sensor is minimally invasive, and cantherefore be used in biomedical sensing.

UK Patent Application No. 2 391 314 describes electrochemical sensorsfor measuring the amount of hydrogen sulphide or thiols in a fluid. Thesensor comprises a precursor and reaction solution which, together withthe hydrogen sulphide or thiols, create a redox reaction. The currentproduced by this redox reaction is dependent upon the concentration ofhydrogen sulphide or thiols. The sensors described in this document arefor use in downhole applications, i.e. to extend down boreholes during adrilling operation. Given the size restrictions on apparatus which mustextend into a borehole, the sensors must be relatively small.

Carbon-based electrode materials have been in use for many decades. Themain forms of carbon in common use are glassy carbon, carbon fibres,carbon black, graphite, carbon paste and carbon epoxy electrode. Carbonis an attractive electrode material as it is relatively chemically inertyet it has a high surface activity and a wide operational potentialwindow (ca. −1.0 V to +1.0 V vs. the saturated calomel electrode inaqueous solution).

However, there remains a need for more robust, reagentless sensors thatcan provide accurate results in hostile environments or “dirty” mediasuch as effluents or sewage. Furthermore, there is a requirement forsensors to be used under various conditions, such as at temperaturesabove room temperature. There is renewed interest in developing sensorscapable of measuring pH accurately at elevated temperatures. The presentinvention aims to address these issues.

SUMMARY OF THE INVENTION

According to the present invention, there is provided an electrode foruse in a electrochemical sensor, said electrode comprising carbonmodified with a chemically sensitive redox active compound which is notanthraquinone, phenanthrenequinone or N,N′-diphenyl-p-phenylenediamine(DPPD).

The present invention also provides a pH sensor comprising:

-   -   a working electrode comprising a chemically sensitive redox        material; and    -   a counter electrode,        wherein the ratio of the surface area of the working electrode        to the surface area of the counter electrode is from 1:10 to        10:1.

The invention further provides a pH sensor comprising:

-   -   a working electrode comprising a chemically sensitive redox        material, and    -   a counter electrode,        wherein the area of the working electrode is from 500 μm² to 0.1        m².

These pH sensors have an advantage over those in the prior art in thatthey are less likely to be fouled or clogged by dirt in the fluid beingmeasured, and accordingly they have a longer lifetime before they needto be replaced.

The electrodes of the present invention may be used in anelectrochemical sensor, and in particular in a pH sensor. The electrodesof the invention and the pH sensors of the invention are preferablysuitable for use in a non-downhole environment.

The invention also provides a method for preparing an electrode for usein an electrochemical sensor, said method comprising modifying carbonwith a chemically sensitive redox active material with the proviso thatthe chemically sensitive redox active material is not anthraquinone,phenanthrenequinone or N,N□-diphenyl-p-phenylenediamine (DPPD).

Finally, the invention further provides a method for preparing anelectrode in situ comprising applying carbon modified with a chemicallysensitive redox active material to the surface of a substrate, whereinthe chemically sensitive redox active material undergoes an irreversiblechemical reaction when subjected to cyclic voltammetry.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is the voltammetric response of 6-nitro chrysene carbonimmobilised on bppg after the development of the reversible system at pH1.0 (0.1M HCl+0.1M KCl) varying the scan rate (25, 50, 100, 200, 300,400, 500, 750, 1000 mV s⁻¹) and FIG. 1B is the corresponding plot ofoxidative peak potential vs. scan rate (insert).

FIG. 2 shows the cyclic voltammograms of PAQ at each pH studied (pH 1,pH 4.6, pH 6.8, pH 9.2 and pH 12). Step potential 2 mV, scan rate 100 mVs⁻¹. FIG. 3A is the base-line corrected oxidative and reductive squarewave voltammograms of DPA at each pH studied (pH 1, pH 4.6, pH 6.8, pH9.2 and pH 12). FIG. 3B is the corresponding plot of oxidative peakpotential vs. pH.

FIG. 4 shows cyclic voltammograms of diphenylamine derivatised carbon inpH 6.8 buffer showing the first, second and tenth scans.

FIG. 5 shows cyclic voltammograms of thionin derivatised carbonparticles immobilised on a bppg electrode in pH 12.0 (0.1M NaOH+0.1MKCl) buffer showing the first and fourth scans.

FIG. 6A shows cyclic voltammograms of 6-nitrochrysene derivatised carbonpowder immobilised on a bppg electrode in pH 9.2 buffer (0.05M sodiumtetraborate+0.1M KCl) showing the first and tenth scans. FIG. 6B: 10 CVscans 9-nitroanthracene derivatised carbon powder immobilised on a bppgelectrode over the reversible systems (see text) at pH 6.8.

FIG. 7 shows cyclic voltammograms of FBK derivatised carbon powderimmobilised on a bppg electrode in pH 4.6 buffer (0.1M acetic acid+0.1Msodium acetate+0.1M KCl) showing the first and second scans.

FIG. 8A cyclic voltammograms of FBK derivatised carbon powderimmobilised on a bppg electrode in pH 4.6 buffer (0.1M acetic acid+0.1Msodium acetate+0.1M KCl) where the potential is cycled around system(II) only (see text) showing the first and tenth scans.

FIG. 8B: CV scans of the same system with varying scan rate (25, 50, 75,100, 200, 300, 400, 500, 600, 700, 800 and 900 mV s⁻¹)

FIG. 9 shows thirty repeated CVs of azobenzene derivatised carbon at pH4.6 showing the effect of sweeping down to very negative potentials.

FIG. 10 shows baseline corrected oxidative SWV voltammograms ofDPA-carbon at pH 4.6 (0.1M acetic acid+0.1M sodium acetate+0.1M KCl)buffer over a temperature range of 25-80° C. in 5° C. increments.

FIG. 11 shows a pH sensor according to the first preferred aspect of theinvention.

FIG. 12 shows an array of pH sensors according to the first preferredaspect of the invention on a single substrate.

FIG. 13 discloses a schematic diagram illustrating the proposed partialintercalation of 4-NBA into localised edge-plane defect sites along thesurface of a MWCNT.

FIG. 14 discloses the general mechanism for the electrochemicalreduction of an aryl nitro moiety illustrated here by nitrobenzene.

FIG. 15A shows twenty consecutive cyclic voltammograms of 4-NBAcarbon inpH 6.8 buffer.

FIG. 15B shows overlaid cyclic voltammograms recorded before and afterreplacing the pH 6.8 solution with fresh solution.

FIG. 15C shows overlaid cyclic voltammograms of 4-NBAcarbon recordedafter formation of the reversible couple corresponding to system II atvarying scan rates (25, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900mVs⁻¹) in pH 6.8 buffer.

FIG. 15D shows the corresponding plot of peak current vs. scan rate.

FIG. 16A shows the 1^(st) cyclic voltammograms (overlaid) of 4-NBAcarbonrecorded in solutions of varying pH (pH 1.0, pH 4.6, pH 6.8, pH 9.2 andpH 12.0).

FIG. 16B shows the corresponding plot of peak potential vs. pH.

FIG. 17A shows overlaid square wave voltammograms (reductive sweep) of4-NBA derivatised MWCNTs recorded in solutions of varying pH (pH 1.0, pH4.6, pH 6.8, pH 9.2 and pH 12.0).

FIG. 17B shows the corresponding plot of peak potential vs. pH.

FIG. 18 is a plot of system I peak area vs. time for 4-NBAcarbon sampledat various times during derivatisation. Error bars show the standarddeviation over 5 samples.

FIG. 19 shows five consecutive cyclic voltammograms of 4-NBA carbon inacetonitrile containing 0.1M LiClO₄.

FIG. 20 depicts a schematic model of 4-NBA partially intercalated intographite showing that solvated Li⁺ cation may come into close contactwith the 4-NBA molecule whereas the solvated NH₄ ⁺ cation may not.

FIG. 21 shows a schematic model of the structure of graphite showing theapproximate dimensions of a 4-NBA molecule and the graphite interlayerspacing for comparison.

FIG. 22A shows a scanning electron microscopy image of MWCNTs modifiedwith 4-NBA.

FIG. 22B shows a scanning electron microscopy image of unmodified“native” MWCNTs.

FIG. 23A shows a high resolution transmission electron microscopy imageof a “bamboo-like” region of a MWCNT.

FIG. 23B shows a high resolution transmission electron microscopy imageof a “hollow tube” region of a MWCNT.

FIG. 24 shows X-ray powder diffractograms comparing unmodified “native”MWCNTs with 4-NBA modified MWCNTs.

FIG. 25 is a schematic diagram showing the regions where the formationof a three-phase boundary may occur when a redox active material such asan organic crystal is immobilised on the surface of an electrode.

FIG. 26 is a schematic diagram showing the regions where the formationof a three-phase boundary may occur when a redox active material such asan organic crystal is mixed with carbon particles and immobilised on thesurface of an electrode.

FIG. 27 is a schematic diagram showing the regions where the formationof a three-phase boundary may occur when a redox active material such asan organic crystal is mixed with carbon nanotubes and immobilised on thesurface of an electrode. For clarity, the electrode surface is notshown.

FIG. 28 is a schematic diagram showing the regions where the formationof a three-phase boundary may occur when a redox active material such asan organic crystal is agglomerated with carbon nanotubes and immobilisedon the surface of an electrode. For clarity, the electrode surface isnot shown.

FIG. 29 is an SEM image of abrasively immobilised nanotubes dispersed onthe surface of a bppg electrode.

FIG. 30 is an SEM image of abrasively immobilised agglomerates ofmulti-walled carbon nanotube and 9,10-phenanthraquinone (MWCNT-PAQagglomerates) on the surface of a bppg electrode.

FIG. 31 is a magnified SEM image of an MWCNT-PAQ agglomerate.

FIG. 32 is a digital image of a bppg electrode after abrasiveimmobilisation of pure PAQ crystals.

FIG. 33 is a digital image of a bppg electrode after abrasiveimmobilisation of a physical mixture of PAQ crystals and multi-walledcarbon nanotubes (MWCNTs).

FIG. 34 is a digital image of a bppg electrode after abrasiveimmobilisation of an MWCNT-PAQ agglomerate.

FIG. 35 is the overlaid cyclic voltammograms recorded from each materialpictured in FIGS. 8, 9 and 10, allowing a comparison of the magnitude ofpeak currents compared to the amount of material visibly present on thesurface of the bppg electrode.

FIG. 36A is a cyclic voltammogram showing 20 repeat cycles (scan rate 50mV s⁻¹) in pH 6.8 buffer of MWCNT-PAQ agglomerates on bppg, and FIG. 36Bis a cyclic voltammogram showing 20 repeat cycles (scan rate 50 mVs⁻¹)in pH 6.8 buffer of agglomerates of multi-walled carbon nanotubes and1,2-napthaquinone (MWCNT-NQ agglomerates) on bppg. A cyclic voltammogramrecorded after replacing the buffer with fresh solution is overlaid ineach case.

FIG. 37A is a cyclic voltammogram at varying scan rates (25-900 mV s⁻¹)recorded in pH 6.8 buffer of MWCNT-PAQ agglomerates on bppg, FIG. 37Bshows the corresponding plots of peak current against rate scan forMWCNT-PAQ agglomerates on bppg. FIG. 37C is a cyclic voltammogram atvarying scan rates (25-900 mVs⁻¹) recorded in pH 6.8 buffer of MWCNT-NQagglomerates on bppg. FIG. 37D shows the corresponding plots of peakcurrent against scan rate for MWCNT-NQ agglomerates on bppg.

FIG. 38 is overlaid oxidative and reductive square wave voltammogramsrecorded in a range of buffers (pH 1.0, pH 4.6, pH 6.8, pH 9.2, pH 12.0)at 20° C. wherein FIG. 38A uses MWCNT-PAQ agglomerates on bppg, and FIG.38B uses MWCNT-NQ agglomerates on bppg.

FIG. 39A is an overlaid square wave voltammogram of MWCNT-PAQagglomerates on bppg, and FIG. 39B is an overlaid square wavevoltammogram of MWCNT-NQ agglomerates on bppg, both in pH 6.8 buffer atvarying temperatures (20, 30, 40, 50, 60 and 70° C.).

FIG. 40 is overlaid oxidative and reductive square wave voltammograms ofMWCNT-NQ agglomerates in a range of buffers (pH 1.0, pH 4.6, pH 6.8, pH9.2 and pH 12.0) at 70° C.

FIG. 41 is a schematic of a print, laminate and cut (PLC) electrode inaccordance with the fourth preferred aspect of the present invention.

FIG. 42 shows a micrographic of the PLC electrode.

FIG. 43 shows the first scan of an uncut electrode (lower plot) and thefirst scan of the same electrode after it has been “cut” (upper plot).

FIG. 44 shows the same data as FIG. 43, with the square wavevoltammagram (SWV) generated from a laminated cut electrode (lowestplot).

DETAILED DESCRIPTION OF THE FIRST PREFERRED ASPECT OF THE INVENTION

The pH sensors and electrodes of the first preferred aspect of thepresent invention are preferably used in a non-downhole environment. Theterm “non-downhole” means that the sensors are used in environmentsother than those down boreholes or other subterranean volumes of liquid.Suitable non-downhole applications include pH measurement inenvironmental, chemical, waste water, industrial and effluentapplications. The term “environmental” includes testing such as inrivers and seas. The term “chemical” includes testing during chemicalprocesses in laboratories or factories. The term “industrial” includeswaste from industrial processes. The term “effluent” includes dischargesof liquid waste, for example sewage. Effluent may be mostly or entirelynon-aqueous. Waste water includes liquid waste from domestic andcommercial properties. Waste water is predominantly aqueous.

In the following description the term “chemically irreversiblebehaviour” means that the compounds concerned react via an irreversiblechemical reaction to form another species.

The compound and the species into which it converts will have differentelectrochemical profiles. The term “chemically reversible behaviour”means that the compounds undergo reversible reactions when used in thesensors of the invention, and accordingly do not convert into otherspecies which cannot convert back into the original compound.

The term “reversible electrochemistry” is used interchangeably with thephrase “electrochemically reversible behaviour”. This means that thecompound can gain and lose electrons repeatedly without itselectrochemical profile varying over time.

The individual components and aspects of the invention will now bedescribed in more detail.

Carbon

The carbon used in the present invention must be capable of beingmodified by the chemically sensitive redox active material. Preferablyit is in the form of carbon powder. It is particularly preferred thatthe carbon is in the form of graphite particles having a mean diameterof from 0.1 to 50 μm, preferably from 1 to 30 μm. Alternatively thecarbon can be present in the form of carbon nanotubes. These are ineffect “rolled up” sheets of graphite.

Conventionally they are either single-walled carbon nanotubes ormulti-walled carbon nanotubes (MWCNTs).

Chemically Sensitive Redox Active Material

The chemically sensitive redox active material may be any organicmaterial capable of undergoing electron loss and gain. Preferably it isa solid phase material. When immobilised onto a substrate, e.g. glassycarbon or a basal plane pyrolytic graphite (bppg) electrode, itundergoes concomitant proton and electron loss/gain onoxidation/reduction.

This material is described as being “chemically sensitive” because itmust show an electrochemical response which is dependent upon thespecies which is to be detected or measured. For example, in order to beof use in a pH sensor, the chemically sensitive redox active materialmust have an electrochemical response which is sensitive to a change inhydrogen ion concentration.

This material need not comprise only one compound, but can insteadcomprise a mixture of different chemically sensitive redox activecompounds.

Preferred chemically sensitive redox active materials are those whichcomprise a) compounds exhibiting chemically and electrochemicallyreversible behaviour, and b) compounds exhibiting chemicallyirreversible behaviour leading to electrochemically reversiblebehaviour. Of the latter category, preferred materials comprise b.1)compounds which form polymers, and b.2) compounds which contain a nitrogroup. Considering each of these groups in turn:

a) Redox Active Materials Comprising Compounds Exhibiting Chemically andElectrochemically Reversible Behaviour

The compounds in these redox active materials produce stable reversiblevoltammetric peaks when they are subjected to both cyclic voltammetryand square-wave voltammetry. They exhibit Nernstian behaviour. When thesensor in which they are incorporated is intended to measure pH, theplot of peak potential against pH for each compound produces a linear,Nernstian response according to the following equation:

$E_{peak} = {E_{formal}^{0} - {\frac{2.3\mspace{14mu} {RTm}}{nF}{pH}}}$

wherein E_(peak) is the peak potential/V, E⁰ _(formal)/V is the formalpotential, R is the universal gas constant/J K⁻¹ mol⁻¹, T is thetemperature/K, F is the Faraday constant/C mol⁻¹, and n and m are thenumber of electrons and protons transferred respectively. In theexamples which follow, n and m are both likely to be equal to two asproposed in scheme 1. By monitoring the peak potential of thesecompounds, the pH can be determined.

Suitable compounds which exhibit chemically and electrochemicallyreversible behaviour include quinones and anthracenes. Compounds whichwill be effective in pH sensors are capable of undergoing a redoxreaction that is reversible and involves the uptake and release ofprotons. The skilled person will be able to determine other compoundswhich will be suitable in this embodiment of the invention.

b) Redox Active Materials Comprising Compounds Exhibiting ChemicallyIrreversible Behaviour Leading to Electrochemically Reversible Behaviour

The compounds in these redox active materials, when subjected to cyclicvoltammetry, undergo an irreversible chemical reaction. The substanceformed after as a result of this irreversible chemical reaction (e.g. adifferent compound or a polymer of the reactant compound) exhibitselectrochemically reversible behaviour similar to the compoundsdescribed in paragraph a) above. The peak potential of these substancesshows a Nernstian response to the species to be measured (the targetanalyte) and these substances can therefore be used in electrochemicalsensors such as pH sensors.

The compounds in this group can be split into a number of subsets,including:

b. 1) Those which Form Polymers

The compounds useful in this aspect of the invention all form polymersvia an irreversible chemical reaction. The resulting polymers have peakpotentials which exhibit a linear, Nernstian response to the species tobe measured when subjected to cyclic voltammetry. Suitable compounds foruse in this aspect of the invention include diphenylamine andphenothiazine dyes, with diphenylamine being most preferred. Suitablephenothiazine dyes include toluidine blue, methylene blue and thionin.Of these, methylene blue and thionin are preferred.

In the prior art, the majority of existing amperometric pH sensors arebased on the pH-switchable permselectivity of thin films and membranes.One such family of conducting polymeric films is based onpolyaniline-like structures formed by electro-oxidative methods. Thepolymerisation of diphenylamine has been carried out electrochemicallyin non-aqueous solvents rather than in solution via chemical means dueto the poor solubility of polydiphenylamine in most solvents. Much ofthe discussion in the prior art has focused on the coupling mechanismfor the formation of these polymers from diphenylamine in non-aqueoussolvents on the surface of gold or platinum electrodes.

However, the present invention differs from the prior art in that thecompounds can be used to modify carbon. For example, they can bephysisorbed onto the surface of carbon particles. When this occurs, thecompounds undergo an oxidative electropolymerisation reaction whilst incontact with aqueous solutions to form polymers, which are themselvessensitive to changes in local pH. To apply this to a particularcompound, when diphenylamine is used to modify carbon, the compound canthen undergo oxidative electropolymerisation whilst in contact withaqueous solutions to form polydiphenylamine, which is itself useful insensing changes in local pH.

In general, the behaviour of compounds according to this aspect of theinvention is as follows. When subjected to cyclic voltammetry, aninitial peak is observed, corresponding to the redox active material inits original form. Gradually this peak disappears as eventually all ofthe redox active material on the surface of the carbon is polymerised. Anew reversible system is then generated, corresponding to the polymericform of the redox active material. The peak corresponding to this systemgrows upon repetitive cycling and eventually stabilises.

By monitoring the peak potentials of both the irreversible peak and thereversible peak, the pH can be monitored. Analysis of the gradient of aplot of peak potential vs. pH for both the irreversible system and thereversible system shows a shift in a linear Nernstian fashion.

Thus, these redox active materials not only provide a robust reagentlesspH sensor over a wide range, but also provide a technique for preparingan electrode in situ.

b.2) Those which Contain a Nitro Group

Such redox active materials are usually aromatic compounds containingone or more carbocyclic rings and/or heterocyclic rings and aresubstituted by at least one nitro group. Suitable carbocyclic compoundsinclude phenyl, anthracene, fluoranthene and the like. Suitableheterocyclic compounds include the carbocyclic compounds having one ormore carbon atoms in the ring replaced by one or more heteroatoms. Theheteroatoms are preferably selected from nitrogen, oxygen and sulphur.Preferably the heterocyclic compounds contain, in the ring, from one tofive heteroatoms, preferably from one to three heteroatoms. Thecompounds should preferably not undergo any other competing chemicaltransformations or reactions which mask their response to the specieswhich is to be measured by the electrochemical sensors of the invention.

Preferred compounds include nitroanthracene, in particular where theanthracene molecule is substituted by the nitro group in the 9 position;nitrochrysene, in particular where the nitro group is in the 6 position;and nitrofluoranthene, in particular where the nitro group is in the 3position. Other compounds which can be used include fast black K (FBK).

The electrochemical reductions of aromatic compounds follow complexmechanistic pathways in both aqueous and non-aqueous media, and at thethree-phase boundary between microdroplets of oils containing nitrogroups, aqueous electrolyte and an electrode. There has been speculationabout the exact mechanism of the reduction of nitro groups, with variousdifferent pathways being suggested depending on the pH of the solution.However, it is now generally agreed that the pathway shown below inScheme 2 below is that followed.

This scheme uses nitrobenzene as an example of such a compound, but thegeneral scheme is the same for the other nitro-containing compounds.

In this mechanism the nitro group undergoes a six-electron, six-protonreduction to form the corresponding aryl amine which is split into twosteps: a four-electron, four-proton reduction to the aryl hydroxylaminevia the nitroso intermediate, and a further two-electron, two-protonreduction to the aryl amine.

As with the compounds which form polymers, these redox active materialsnot only provide a robust reagentless pH sensor over a wide range, butalso provide a technique for preparing an electrode in situ.

Chemically Insensitive Redox Active Materials

In addition to the chemically sensitive redox active material, theinvention may also employ chemically insensitive redox active materials.Thus, a chemically insensitive redox active material may be present onthe same substrate as the chemically sensitive redox active material.

A chemically insensitive redox active material may also be present on aseparate electrode used in an electrochemical sensor according to theinvention.

The chemically insensitive materials may be used to modify carbon, aswith the chemically sensitive materials. Alternatively, these materialscan be applied directly to the substrate (to bppg, for example). If thematerial is to be screen printed, then a binder and thinner would alsobe required.

The chemically insensitive redox active materials are, like thechemically sensitive redox active materials, capable of undergoingrepeated electron loss and gain. However, the electrochemical responseof these compounds is not dependent on the concentration of the speciesto be measured by the sensor. For example, if the sensor is a pH sensor,then the electrochemical response of the chemically insensitive redoxactive material will be insensitive to a change in concentration ofhydrogen ions, and will therefore be insensitive to a change in pH.

The choice of the chemically insensitive redox compounds will clearlyalso depend upon the species which the electrochemical sensor is todetect. In the case of pH sensors, suitable compounds include insolubleferricyanide or ferrocyanide salts, octacyanomolybdate (IV) salts, orpolymers such as polyvinylferrocene. These compounds act as referencematerials, which generate their own, reference signals when subjected tocyclic voltammetry. The difference in potential between the peaks of thechemically sensitive redox compounds and the chemically insensitiveredox compounds can be used to improve the accuracy of the sensor, asdiscussed in U.S. Pat. No. 5,223,117.

Substrate

The substrate onto which is applied the modified carbon may be anysubstrate conventionally used in the manufacture of electrodes. Forexample, the substrate may be a basal plane pyrolytic graphite (bppg)electrode or glassy carbon, metal electrodes such as gold or platinum,or optically transparent electrodes such as those comprising ITO. Thesubstrate preferably has good electrical contact with the modifiedcarbon, and also has a surface such that good coverage with the modifiedcarbon can be achieved.

The modified carbon may be applied by any known procedure. Inparticular, screen-printing is a suitable conventional technique.

Working Electrode

The size of the working electrodes of the present invention sensor issuch that the surface area is preferably from 10 μm² to 0.1 m², morepreferably from 50 μm² to 0.1 m², and more preferably from 500 μm² to0.1 m²

Another relevant parameter when considering the electrodes of thepresent invention is the ratio of the surface area of the workingelectrode to the surface area of the counter electrode. This ispreferably from 1:10 to 10:1, more preferably from 1:5 to 3:1.

In contrast to the electrodes of the present invention, those disclosedin the prior art tend to have a much smaller surface area of the workingelectrode and/or have a counter electrode which is substantially largerin size than the working electrode.

The electrodes of the present invention have the advantage that they areless likely to be fouled by the substances they are being used toanalyse. The working life of the sensors is therefore lengthened beforethe electrode needs to be replaced.

Furthermore, the electrodes of the invention can be made cheaply and canbe readily disposed of. While being larger than those discussed in theprior art, they are still small enough to be easily carried and can thusbe used in portable electrochemical sensors.

The working electrodes of the present invention may comprise one or moreareas of redox active material on the same substrate. These areas arepreferably separated from each other, and may thus appear as “spots” onthe surface of the substrate. At least one area will comprise carbonmodified with a chemically sensitive redox active material. The otherarea or areas may comprise chemically sensitive redox active materialsor chemically insensitive redox active materials.

According to one embodiment, the working electrode comprises twoseparate areas, one of which comprises carbon modified with a chemicallysensitive redox active material, the other of which comprises achemically insensitive redox active material. According to anotherembodiment, the working electrode comprises four separate areas, two ofwhich comprise chemically sensitive redox active materials, the othertwo of which comprise chemically insensitive redox active materials. Oneadvantage of having a number of separate areas of material is that anumber of electrodes can be present on one sensor, or a number ofdifferent materials can be deposited on different areas of one workingelectrode. The result of this is that the accuracy and sensitivity ofthe sensor can be increased

pH Sensor

The structure of the sensor will depend upon its final application, andhence depends upon the substance which is to be measured and theenvironment in which measurement will take place. Known sensorstructures may be employed in conjunction with the agglomerates andelectrodes described herein.

Exemplary sensors may have a two or three terminal arrangement. Thus,they may comprise a working electrode of the invention and a combinedcounter and reference electrode, or a working electrode, counterelectrode and a reference electrode. The reference electrode and counterelectrode can be any conventional electrodes known in the art, such assilver electrodes, calomel electrodes or standard hydrogen electrodes.The reference electrode can also be provided by the chemicallyinsensitive redox active material described earlier.

The materials used in the sensor depend upon which species the sensor isintended to measure and the environment in which the sensor is to beused. In order to modify the sensor to be sensitive to a differentspecies it is simply required for the skilled person to substitute theredox active material with a different redox active material sensitiveto the species which is to be measured.

An exemplary pH sensor according to the invention is shown in FIG. 11.The substrate 1 bears the electrodes 2, 3 and 4. The counter electrode 2and the reference electrode 3 both comprise silver. In this Figure theworking electrode comprises carbon derivatised withN,N′-diphenyl-p-phenylenediamine (DPPD), with the electrode containing10% by mass of DPPD. The pH sensor is connected to equipment to measurecyclic voltammetry via the three terminals 5.

FIG. 12 shows another embodiment of the invention in which fourdifferent sensors are present on one base. The sensors are produced byscreen printing, and can be cut into individual sensor strips whenrequired. The numerals used in this Figure correspond to those used inFIG. 11 above.

Modification Method

The preferred methods for modifying the carbon used in the presentinvention are

-   -   a) homogeneous chemical derivatisation with the chemically        sensitive redox active material;    -   b) derivatisation via physical adsorption of the chemically        sensitive redox active material; and    -   c) physical mixing with the chemically sensitive active material        and a binder.

Homogeneous chemical derivatisation describes a method wherein thecarbon and chemically sensitive redox active material are combined in asolvent the presence of a reductant (e.g. hypophosphorous acid) in orderto cause chemical bonding of the two species. The term “homogeneous”refers to the fact that all of the reductant (and the reductant only) isin the solution phase. The carbon is dispersed in the solvent while thereductant and the chemically sensitive redox active material (or aprecursor thereof) are dissolved in solution. The chemical reactionoccurs solely in the solution phase to generate a species which thenbonds to the surface of the carbon.

Derivatisation via physical adsorption means that the carbon andchemically sensitive redox active material are combined in such a way asto cause the latter to become physically adsorbed onto the surface ofthe carbon.

The two processes above differ in that, in the case of physicaladsorption, the skilled person relies on relative hydrophobicities toinduce derivatisation of the carbon surface. In chemical derivatisationan actual chemical bond is formed between the carbon and the chemicallysensitive redox active material.

The physical mixing option simply requires the carbon and chemicallysensitive redox active material to be mechanically mixed together in thepresence of a binder. This allows the carbon and redox active materialto be associated with one another without forming chemical bonds. Thechoice of binder depends on the conditions to which the electrode willbe subjected. Conventional binders and thinners, such as those employedin the screen-printing industry, are possible candidates.

Examples of the First Preferred Aspect of the Invention Reagents andEquipment

All reagents were obtained from Aldrich (except for methylene blue andthionin which were obtained from British Drug House Chemicals andpotassium chloride which was supplied by Riedel de Hafn) and were of thehighest grade available and used without further purification. Allaqueous solutions were prepared using deionised water from an Elgastat(Elga, UK) UHQ grade water system with a resistivity of not less than 18MΩ·cm. All measurements were made after degassing the solution with pureN₂ gas (BOC gases, Guildford, Surrey, UK) for 30 minutes and unlessstated otherwise results were recorded at a temperature of 22±2° C.

Solutions of known pH in the range pH 1 to 12 were made up in de-ionisedwater as follows: pH 1, 0.1M HCl; pH 4.6, 0.1M acetic acid+0.1M sodiumacetate; pH 6.8, 0.025M Na₂HPO₄+0.025M KH₂PO₄; pH 9.2, 0.05M disodiumtetraborate; pH 12, 0.01M sodium hydroxide. These solutions contained inaddition 0.1M KCl as additional supporting electrolyte.

pH measurements were performed on each freshly made solution to ensureit had the correct pH using a Jenway 3030 pH meter.

Electrochemical measurements were recorded using a giAutolab computercontrolled potentiostat (Ecochemie, Netherlands) with a standardthree-electrode configuration. All room-temperature experiments werecarried out in a cell of volume 30 cm³. High-temperature voltammetry(30-70° C.) was undertaken using a double-walled glass cell of volume 25cm³ thermostatted to the desired temperature through circulation ofwater from a heated water bath. In all cases a basal plane pyrolyticgraphite (bppg, 0.20 cm², Le Carbone Ltd., Sussex, UK) electrode actedas the working electrode (see below). A platinum rod acted as thecounter electrode, and a saturated calomel electrode as the referenceelectrode (SCE, Radiometer, Copenhagen) completed the cell assembly.

Unless stated otherwise cyclic voltammograms were recorded using thefollowing parameters: step potential 2 mV, scan rate 100 mV s⁻¹. Squarewave voltammetric parameters were as follows: frequency 12.5 Hz, steppotential 2 mV and amplitude 5 mV.

Scanning electron microscopy (SEM) was conducted using a Cambridgestereoscan electron microscope at a magnification of 83×. Initialcharacterisation of the size of the carbon particles was carried outusing scanning electron microscopy (SEM). This involved attaching thecarbon particles to a strip of conducting sticky tape, from which theSEM image was taken. Analysis of the image revealed that the carbonparticles had a mean diameter of 1.5 μm, consistent with that stated bythe manufacturer (Aldrich, graphite powder, 1-2 μm, synthetic).

Example 1 Carbon Powder Derivatisation Methods

a) Derivatisation Via Physical Adsorption:

Physical adsorption onto carbon powder was carried out by mixing 2 g ofcarbon powder with 25 cm³ 0.1M HCl+0.1M KCl and 10 cm³ of a 10 mMsolution in acetone of one of the following compounds: anthracene,azobenzene (AB), diphenylamine, 9,10-diphenylanthracene (DPA),1,3-diphenyl guanidine, fluorescein, methylene blue, 3-nitrofluoranthene(3-NF), 6-nitrochrysene (6-NC), 9-nitroanthracene (9-NA),9,10-phenanthraquinone (PAQ) or triphenylamine. The reaction mixture wasstirred continuously for 2 hours in a beaker and then filtered by watersuction after which it was washed with distilled water to remove theacid and salt. It was then air-dried by placing inside a fume hood for12 hours and finally stored in an airtight container.

b) Homogeneous Chemical Derivatisation

Initially 2 g of carbon powder was mixed with a 10 cm³ solutioncontaining 5 mM Fast Black K(2,5-dimethoxy-4-[(4-nitrophenyl)azo]benzenediazonium chloride; FBK), towhich 50 cm³ hypophosphorous acid (H₃PO₂, 50%; Aldrich) was added. Thereaction mixture was then left to stand at 5° C. for 30 minutes withstirring every ten minutes, after which the solution was filtered bywater suction in order to remove any unreacted species from the carbon.Further washing with deionised water was carried out to remove anyremaining acid and finally with acetonitrile to remove any unreacteddiazonium salt from the mixture. The carbon particles were thenair-dried by placing inside a fume hood for a period of 12 hours afterwhich they were stored in an airtight container.

Lifetimes of the Derivatised Carbon Powders:

Each compound derivatised using one of the methods given above andstored in an airtight container was studied over a period of severalmonths and was found to produce stable voltammograms after this periodof time had elapsed. This shows that there is little or no desorptionfrom the carbon particle surface and that the derivatised carbon powdersare stable over time

Example 2 Immobilisation of the Derivatised Carbon onto a Substrate

The newly derivatised carbon powders were characterised by abrasiveimmobilisation onto the surface of a bppg electrode prior tocharacterisation. This was done by initially polishing the electrode onglass polishing paper (H00/240) after which they were polished onsilicon carbide paper (P1000C) for smoothness. The derivatised carbonwas then mechanically immobilised onto the bppg electrode by gentlyrubbing the electrode surface on a fine filter paper (Whatman)containing the functionalised carbon. It is worth noting that in thecase of 3-nitrofluoranthene, 6-nitrochrysene and 9-nitroanthracenederivatised carbon powders, the derivatised carbon was immobilised ontothe basal plane at the beginning of each set of experiments as theelectro-reduction of the nitro group is chemically irreversible andhence the signal is lost after the first initial scan (see below).

Characterisation Protocol:

In order to verify that each compound studied was attached to the carbonparticles, either through physical adsorption or via a covalent bonddepending on the derivatisation method, the following protocol wascarried out using cyclic voltammetry over the entire pH range studied(pH 1-12) on each newly derivatised carbon immobilised onto a bppgelectrode. First ten repetitive scans (not shown) from +1.0 V to −1.0 Vwere typically conducted to ensure the stability of the species. In eachcase an electrochemically reversible system could be observed whichrapidly stabilised to give a nearly symmetrical wave shape with aseparation of ca 20 mV between the oxidative and reductive peaks whichis close to the ideal zero peak to peak separation for an immobilisedspecies. Next the electrolyte solution was replaced with fresh solutionand the voltammetric response recorded. The corresponding voltammetricresponse (not shown) was found to overlay the last scan therebyconfirming that the electroactive species remains on the electrodesurface. Finally the scan rate was varied and a plot of peak current vs.scan rate was found to be linear, consistent with a surface boundspecies.

Together these tests each confirm that a particular compound studied isattached to the surface of the carbon particles. By way of an exampleFIG. 1A shows the voltammetric response of 6-nitrochrysene physicallyderivatised carbon with varying scan rate (25-1000 mV s⁻¹) at pH 1.0(0.1M HCl+0.1M KCl) after development of the reversible system (seebelow). FIG. 1B is a plot of the corresponding peak current against scanrate which is linear as expected for a surface bound species.

All of the compounds studied were found to be immobilised and stablebetween pH 1-12 at room temperature except for thionin and methyleneblue which were both found to slowly desorb upon repetitive cycling atpHs greater than 6.8 and 4.6 respectively. Fluorescein, 1,3-diphenylguanidine and triphenylamine produced very poorly defined voltammetricwaves at any pH and as such no further analysis was performed on them.

Example 3 Voltammetric Response of the Derivatised Carbons at 22° C.From pH 1 to 12

The response of the derivatised carbons at each pH was first studiedindividually using cyclic voltammetry (CV) and then using square wavevoltammetry (SWV). SWV was utilised as the electrochemical probe of thesystem as it has significant advantages to conventional CV, providingwell-defined voltammetric peaks in a single sweep due to thereversibility of each redox system studied. The corresponding cyclicvoltammograms and square wave voltammograms were recorded in a range ofpH solutions (pH 1.0, 0.1M HCl+0.1M KCl; pH 4.6, 0.1M acetic acid+0.1Msodium acetate+0.1M KCl; pH 6.8, 0.025M Na₂HPO₄+0.025M KH₂PO₄+0.1M KCl;pH 9.2, 0.05M disodium tetraborate+0.1M KCl; pH 12, 0.01M KOH+0.1M KCl).The voltammetric behaviour of compounds of the invention can be groupedinto three types: (1) chemically and electrochemically reversiblebehaviour, (2) chemically irreversible leading to electrochemicallyreversible systems involving the formation of polymeric species and (3)chemically irreversible leading to electrochemically reversible systemsinvolving nitro containing compounds.

Example 3.1: Compounds displaying chemically and electrochemicallyreversible behaviour The voltammetric response of graphite powderderivatised with PAQ and DPA according to the method in Example 1 wasmeasured. FIG. 2 shows the overlaid cyclic voltammetric responses of PAQmeasured over the pH range 1-12. The peak shapes are nearly symmetricalwith a slight separation of ca 20 mV between oxidative and reductivepeaks at each pH. A slight shoulder at higher potential can be observedon each peak which was not observed with either DPA or anthracene. Thisis analogous to the voltammetry observed when anthraquinone isderivatised onto carbon powder and can be tentatively attributed tointermediate reduction/oxidation of the quinone/semi-quinone speciesrespectively.

FIG. 3A shows the overlaid oxidative and reductive SWV response of DPAover the pH range 1-12 and FIG. 3B shows the corresponding plot of peakpotential against pH. This plot clearly shows a linear response to pHwith a gradient of 61 mV/pH unit which is in excellent agreement withtheory (equation 1). A comparison of the experimentally obtainedpotential shifts with pH for each compound with theory is given in Table1 for each compound in this class.

TABLE 1 Comparison between theoretically calculated shift in peakpotential with pH (58.1 mV/pH unit, equation 1) and experimentallydetermined shifts of peak potential with pH for anthracene, DPA and PAQtaken from oxidative SWV scans at 22 ± 2° C. Experimental Shift ± 2Compound (mV/pH unit) Anthracene 57.5 9,10-Diphenylanthracene 61.69,10-Phenanthraquinone 56.3

Example 3.2 Compounds Displaying Chemically Irreversible BehaviourLeading to Electrochemically Reversible Behaviour—Compounds which FormPolymers

Cyclic voltammetry of carbon powder derivatised with diphenylaminerevealed that upon first scanning in an oxidative direction a largeelectrochemically irreversible wave is observed at ca +0.45 V vs. SCE atpH 6.8 (FIG. 4). Upon reversing the scan direction at +1.0 V a new waveis observed at ca +0.03 V vs. SCE which upon repetitive cycling grew togive a stable, reversible redox system, whilst the large peak at +0.45 Vdied away after 4 cycles. This behaviour is analogous to that reportedin the literature for electropolymerisation of diphenylamine in solutionexcept that in this case the polymerisation occurs for diphenylaminephysisorbed onto the surface of carbon particles in contact with anaqueous solution. The large electrochemically irreversible peak labelled(I) in FIG. 4 can be attributed to the oxidation of diphenylamine to itscorresponding radical cation and subsequent polymerisation via amechanism involving concomitant proton loss and gain as shown in scheme3.

Upon repetitive cycling peak (I) disappears as eventually all thediphenylamine on the surface of the carbon is polymerised. A newreversible system, labelled as (II) in FIG. 4, grows upon repetitivecycling and stabilises. This can be tentatively attributed to the redoxresponse of the polydiphenylamine involving subsequentoxidation/reduction of the imine linkages in the polymer structure andsubsequent proton loss/gain (scheme 3). It is worth noting that innon-aqueous media the polymerisation is thought to begin with thedimerisation of diphenylamine to form diphenylbenzidine and twocorresponding reversible waves were reported at a higher potential thanthose corresponding to the polymeric species. In the present case twosmall reversible waves are observable at ca +0.25 V for the first fewscans.

These are tentatively attributed to oxidation/reduction ofdiphenylbenzidine, but these too die away and by the tenth scan they canhardly be observed as the dimer is further polymerised to formpolydiphenylamine (scheme 3).

It was found that this voltammetric response was characteristic ofdiphenylamine derivatised carbon at each pH studied from pH 1-12 andthat the peak potentials of both peak (I) and peaks (II) shifted in anegative direction as predicted from equation (1). Analysis of thegradient of a plot of peak potential for both the irreversible system(1) and the reversible system (II) against pH (not shown) found thateach system shifted by 56 mV/pH unit and 66 mV/pH unit respectively in alinear, Nernstian fashion over the entire pH range. This suggests thatcarbon particles derivatised with diphenylamine which subsequentlyundergoes electropolymerisation to form polydiphenylamine provide notonly a robust reagentless pH sensor over the pH range 1-12, but also anovel technique to prepare an electrode in situ in an aqueousenvironment.

Example 3.3 Compounds Displaying Chemically Irreversible BehaviourLeading to Electrochemically Reversible Behaviour—Compounds which FormPolymers

Another class of polymeric films is derived from phenothiazine dyes suchas toluidine blue, and importantly in the present context, methyleneblue and thionin. These molecules are often used as mediators inamperometric sensors coated in Nafion films that detect biologicallyactive molecules and enzymes such as nicotinamide adenine dinucleotide(NADH) and β-d-glucose. The redox properties of methylene blue andthionin derivatised carbon particles are more complicated than that ofdiphenylamine-carbon and very much dependent on pH. In solution thenumber of electrons transferred (n) for both methylene blue and thioninis reported to always be equal to two but the number of protonstransferred (m) is reported to vary with pH such that m=3 at pH<5.4, m=2at pH 5.4<6.0 and m=1 at pH>6.0.

During the characterisation of both methylene blue and thioninderivatised carbon particles using cyclic voltammetry, it was observedthat below a certain oxidising potential (which depended on pH butvaried form +1.2 V at pH 1.0, +1.0 at pH 4.6, +0.65 V at pH 9.2 to +0.4Vat pH 12.0 vs. SCE) reversible waves were observed corresponding to theoxidation/reduction of the monomeric species. If the potential was sweptbeyond this oxidising potential a new wave was observed which has beendescribed in the literature as corresponding to the oxidativeelectropolymerisation methylene blue or thionin respectively (FIG. 5).Upon reversal of the scan direction the peaks corresponding to thereduction of the monomeric species were absent. On repetitive cycling abroad, low, undefined wave at a potential ca 0.2 V more positive thanthat corresponding to the monomer was observed which is analogous tothat reported in the literature and has been attributed to the redoxproperties of the polymer (FIG. 5). Below pH 4.6 the variation of peakpotential of the monomeric species with pH was found to be 86 mV/pH unitand 83 mV/pH unit for methylene blue and thionin respectively; above pH6.8 the shift in peak potential with pH was found to be 33 mV/pH unitand 33 mV/pH. This is analogous to the behaviour reported in theliterature for both species in solution. This demonstrates that carbonparticles can successfully be modified by methylene blue and thionin,and that the resulting modified particles are useful in electrochemicalsensors.

Example 3.4 Compounds Displaying Chemically Irreversible BehaviourLeading to Electrochemically Reversible Behaviour—Compounds whichContain Nitro Groups

The behaviour of 9-NA, 6-NC and 3-NF derivatised carbon powder can begenerically characterised by discussing the voltammetry observed for6-NC. Where differences in behaviour between compounds arise they willbe discussed.

Upon first scanning the freshly immobilised 6-NC in a reductivedirection from +1.0 V to −1.0 V vs. SCE at pH 9.2 a large, reductivepeak was observed at −0.75 V (labelled as (I) in FIG. 6A). Uponreversing the scan direction at −1.0 V no reverse peak was seen but anew oxidative peak (labelled as (II) in FIG. 6A) was observed at ca−0.25 V and a low, broad oxidative wave was also observed at ca 0.25 V.Upon repetitive cycling the electrochemically reversible system at −0.25V stabilised while the electrochemically irreversible reductive wave at−0.75 V rapidly died away (FIG. 6A). In the case of 9-NA a furtherreversible wave at ca −0.55 V also grew with repetitive cycles labelledas (III) in FIG. 6B. The reductive wave in the system at −0.25 V has apronounced shoulder on it, this will be discussed below. Both waves Iand II (and III in the case of 9-NA) were found to be present at each pHin the range 1 to 12 and shifted in a Nernstian, linear fashion (where nis equal to m in equation (1) and is likely to be equal to four for thesystem labelled (I) and two for system (II)) for all three compounds9-NA, 6-NC and 3-NF. Table 2 details the peak potentials of each systemI and II for each compound studied at pH 6.8 for comparison while Table3 details the shift of each peak with pH for each compound studied.

TABLE 2 A comparison of the peak potentials of system (I) correspondingto the six-electron, six-proton nitro group reduction and system (II)corresponding to the aryl hydroxylamine/aryl nitroso redox system for9-NA, 6-NC and 3-NF at pH 6.8 System (II) System (II) System (I) PeakOxidative Peak Reductive Peak Compound Potential/V Potential/VPotential/V 9-Nitroanthracene −0.722 −0.402 −0.428 6-Nitrochrysene−0.654 −0.210 −0.254 3-Nitrofluoranthene −0.785 −0.107 −0.150

TABLE 3 A comparison between theoretically calculated shift in peakpotential with pH (58.1 mV/pH unit, equation 1) and experimentallydetermined shifts of peak potential with pH for system (I) and system(II) for 9-NA, 6-NC and 3-NF taken from oxidative SWV scans at 22 ± 2°C. Experimental Shift ± 2 (mV/pH unit) Compound System (I) System (II)9-NA 54.3 53.2 6-NC 53.5 52.2 3-NF 56.4 61.3

The origin of each wave will be discussed in turn but first it is worthreiterating the fact that for every compound studied thecharacterisation protocol discussed above was carried out on the systemlabelled (II) after several scans had been performed to stabilise thesystem. In every case the results of all three tests (many repeat scansgiving a stable symmetric wave, replacement of the buffer solution withfresh solution and a linear relationship between peak current and scanrate) described in the protocol above confirmed that each compound wasattached to the surface of the carbon particles at each pH studied frompH 1 to pH 12.

Comparison with the literature reveals that the voltammetric response ofall the compounds studied which is described above and shown in FIG. 6Aand FIG. 6B is characteristic of the electrochemical reduction of anaromatic molecule containing a nitro group and is consistent with thegeneral mechanism shown in scheme 2.

By analogy with the reduction of nitrobenzene peak (I) corresponds tothe four-electron, four-proton reduction of the nitro moiety to thecorresponding arylhydroxylamine, which involves a two-electron,two-proton chemically irreversible reduction followed by a furthertwo-electron, two-proton step.

Upon subsequent cycles the irreversible system at ca 0 V (pH 6.8)labelled as “polymerisation” in FIG. 6A can be tentatively ascribed tothe formation of oligomers by the electro-oxidation of the aryl aminemoiety (formed by sweeping the potential past system (I) and furtherreducing the arylhydroxylamine to the corresponding aryl amine) to itscorresponding radical cation and subsequent polymerisation. This wavealso rapidly dies away as all remaining aryl amine species on thesurface of the carbon is polymerised to form what is apparently anelectro-inactive polymer.

The reversible system labelled as (II) in FIG. 6A grows and stabilisesafter 10 scans at each pH. Again this is characteristic of thevoltammetry reported in the literature and can be attributed to thechemically and electrochemically reversible two-electron, two-protonoxidation/reduction of the aryl hydroxylamine/aryl nitroso moieties.This system remains stable as long as the potential is not swept to veryreducing values (ca −1.2 V at pH 6.8) whereupon the peaks graduallydecrease due to some of the aryl hydroxylamine being further reducedirreversibly to the corresponding amine. A pronounced shoulder isobserved at some pHs on this system, particularly in the case of 9-NAand this may possibly be due to intermediate oxidation/reduction of thearyl hydroxylamine/aryl nitro moiety.

In the case of 9-NA, the system labelled as (III) in FIG. 6B at a morenegative potential than the reduction of the nitroso to hydroxylaminemoiety is not characteristic of a nitro reduction. However, comparisonof the voltammetric behaviour of anthracene discussed above may reveal aclue as to its identity. Comparison of the peak potentials of system(III) and its shift with pH match that of the reversible system observedin the voltammetry of anthracene which undergoes a two-electron,two-proton ring reduction at the 9 and 10 positions shown above inscheme 1.

A two-electron, two-proton reduction at the 9 and 10 position in 9-NA isstill possible, although the presence of the electron withdrawing nitrogroup may affect the redox potential slightly. Furthermore, it was alsoobserved that as the pH was increased from pH 1 to pH 6.8 that themagnitude of the peak current also decreased corresponding to a decreasein the local proton concentration.

Example 3.5 Compounds Displaying Chemically Irreversible BehaviourLeading to Electrochemically Reversible Behaviour—Compounds whichContain Nitro Groups

The voltammetry of FBK(2,5-dimethoxy-4-[(4-nitrophenyl)azo]benzenediazonium chloride)derivatised carbon was also investigated. In this compound the reductionof the nitro group is further complicated by the presence of an azolinkage. Initially a reductive scan was performed using cyclicvoltammetry at each pH. Two irreversible peaks were observed (FIG. 7),the first at higher potential (labelled as (II) in FIG. 7) is as yetunidentified (see below) while the latter at more negative potential ischaracteristic of the now familiar four-electron, four-proton reductionof the nitro group (labelled as (I) in FIG. 7). However the voltammetryof FBK differs from the compounds discussed above because on reversingthe scan direction at −1.0 V no reverse peak was observed for the nitroreduction as expected, but no new oxidative peaks corresponding to thehydroxylamine moiety were observed (although there is again someevidence of possible polymerisation due to the amine being oxidised toits radical cation as a low, broad peak is observed above 0 V with theexact potential dependant on pH.) Upon subsequent repeat cycles nopeaks, either reductive or oxidative are observed at any pH. However,each system, (I) and (II), was found to shift linearly and in aNernstian fashion with pH. The nitro system shifted by 57 mV/pH unitwhile system (II) shifted by 61 mV/pH unit. In order to understand theelectrochemistry further experiments were undertaken using CV where thepotential was swept in a negative direction as far as the firstunidentified system where upon the scan direction was reversed justafter a peak had been observed. It was found that this produced a stablereversible system which, when the characterisation protocol wasperformed upon it, confirmed that the FBK was derivatised onto thecarbon particles consistent with other studies of diazonium saltsderivatised onto carbon through a chemical bond. FIG. 8A shows thisreversible system at pH 4.6 and FIG. 8B shows the cyclic voltammogramswith varying scan rate used in the characterisation protocol. It is onlywhen the potential is swept beyond system (II) and the nitro reductioncorresponding to (I) occurs that all subsequent signals in repeat scansare lost. Having confirmed that the FBK was immobilised onto the carbonsurface and was not desorbing, another explanation for this behaviourwas sought.

Azobenzene was then derivatised onto carbon particles, and it wasverified that it was indeed physisorbed onto the carbon surface usingthe characterisation protocol described above. Cyclic voltammetry ateach pH was recorded. FIG. 9 shows the results of thirty cycles forazobenzene-carbon at pH 4.6. A reversible system is observed with a peakseparation of 130 mV at ca −0.3 V which is close to the potentialobserved for system (II). Furthermore it can be seen that although theazobenzene was physisorbed onto the surface of the carbon and was notobserved to desorb at any pH, if the potential is swept to very negativepotentials of ca −1.0 V vs. SCE the azobenzene peak is seen to graduallydie away. One possible explanation for the large peak separation is thatprotonation effects influence the redox kinetics of adsorbed films ofazobenzene such that the reaction kinetics are sluggish andquasi-reversible when compared to azobenzene in solution where they takeon more reversible character. At pH 1.0 no reverse (oxidative) peak isobserved implying that the reduced form of the azo moiety is eitherirreversibly protonated, or the protonation induces cleavage of the azolinkage. This cleavage is also a likely explanation for the gradual lossof any voltammetric signal from the azobenzene-carbon if the potentialis scanned to very negative potentials as the N—N bond may be furtherreduced.

Given these results, one hypothesis that explains the behaviour of FBKcarbon is given in scheme 4.

Initially scanning in a negative direction from +1.0 V vs. SCE first theazo linkage is reduced in a two-electron, two-proton step to thecorresponding hydrazo form which gives a corresponding Nernstian shiftin peak potential with pH as is observed experimentally. If thepotential is then reversed the corresponding oxidative process occursand the system behaves reversibly and is stable over many scans.However, if the potential continues to be swept to more reducing valuesthen the nitro group is reduced which also leads to the hydrazo-linkbeing cleaved due to nitro reduction occurring at such negativepotentials. Upon reversal of the scan direction no oxidation peakscorresponding to either the nitro group (as expected) or the azo linkage(because it has been cleaved) are observed. However a large broad wavethat is characteristic of amine polymerisation is observed between 0.0to +0.4V depending on pH. After which no further redox processes areobserved in any of the repeated voltammograms. This hypothesis issupported by the mechanistic studies of Heyrovsky et al. whosepolarograms are consistent with a mechanism involving reduction of anitro group with subsequent cleavage of a hydrazo-linkage. Furthermorethe peak area of the nitro reduction peak was always found to besignificantly greater than six times the area of the reduction peakcorresponding to system (II), which by itself is not proof that thismechanism is correct but certainly provides further support in favour ofhydrazo cleavage occurring after the nitro group reduction.

It has thus been demonstrated that despite the complicated mechanisms,product interference and other substituent group interactions when nitrocompounds in accordance with the invention are reduced, a large andclearly resolved irreversible peak corresponding to the four-electron,four-proton reduction of the NO₂ moiety can be observed. The peakpotential of this peak shifts in a linear Nernstian fashion with pH asdetailed in Table 3. These compounds derivatised onto carbon powdertherefore present ideal candidates from which to manufacture so-called“single-shot” disposable reagentless pH sensors for use in environmentswhere a disposable sensor may be preferred to a reusable one, such as insewage and other unpleasant effluents.

Example 4 pH Tests at Elevated Temperatures

In this example, the response of four compounds to pH at temperaturesranging from 20° C. to 70° C. was studied. The experimental resultsobtained were varied with those predicted theoretically using equation1.

It can be seen from equation (1) that as the temperature is increased,so too does the gradient of a plot of peak potential against pH. Afurther point to consider is how the pH of a solution will vary withtemperature as the dissociation constants of the components of thebuffer solution vary as the temperature is changed. Therefore theresponse of PAQ, DPA, anthracene and 9-NA to pH at elevated temperatureswas studied using four IUPAC buffers (pH 1.5, 0.1M potassiumtetraoxalate +0.1M KCl; pH 4.6, 0.1M acetic acid+0.1M sodiumacetate+0.1M KCl; pH 6.8, 0.025M Na₂HPO4+0.025M KH₂PO₄+0.1M KCl; pH 9.2,0.05M disodium tetra borate+0.1M KCl) which have a known pH at eachtemperature studied.

FIG. 10 shows the effect of temperature on the SWV voltammetry of DPA atpH 4.6 showing that elevated temperatures produce enhanced peakcurrents. It is also worth noting that the peak potential shifts in anegative direction with increasing temperature as predicted by equation(1). This behaviour is characteristic of all the compounds selected forinvestigation at high-temperature. Table 4 details the shift of eachcompound with pH at each temperature and compares them to thetheoretical predictions of equation (1). Good agreement is found overthe entire temperature and pH range between theory and experiment thusshowing that carbon powders derivatised with a variety of differentcompounds can be used as reagentless pH sensors from pH 1-9 at elevatedtemperatures up to 70° C.

TABLE 4 A comparison between theoretically calculated (equation 1) andexperimentally determined shifts of peak potential with pH foranthracene, DPA and PAQ taken from oxidative SWV and 9-NA (nitroreduction wave) taken from CV voltammograms over the temperature range20-70° C. Theoretical Temperature Shift Experimental Shift ± 2 (mV/pHunit) (° C.) (mV/pH unit) Anthracene DPA PAQ 9-NA 20 58.1 57.5 61.6 56.352.8 30 60.1 64.8 59.2 55.3 50.0 40 62.1 66.1 60.6 57.2 57.8 50 64.165.3 61.4 61.2 53.7 60 66.1 65.2 62.2 61.2 52.2 70 68.1 65.9 61.2 62.060.2

It will be apparent to those skilled in the art that modifications maybe made to the invention as described above without departing from thescope of the claims below.

DETAILED DESCRIPTION OF THE SECOND PREFERRED ASPECT OF THE INVENTION

The second preferred aspect of the present invention is concerned withthe use of composition comprising carbon and a compound which is anitrobenzene derivative of formula (I):

wherein

-   -   R¹ represents a group of formula —Y or —X—Y wherein Y is        selected from hydrogen, hydroxy, C₁₋₄ alkyl and —NR³R⁴ wherein        R³ and R⁴ are the same or different and are selected from        hydrogen, hydroxy, C₁₋₄ alkyl and C₁₋₄ alkoxy, and wherein X        represents a group of formula —(CRR⁶)_(n)— wherein n is 0 or an        integer from 1 to 4 and R⁵ and R⁶ are the same or different and        are selected from hydrogen, hydroxy, C₁₋₄ alkyl, C₁₋₄ alkoxy or        R⁵ and R⁶ together form a group of formula ═O or ═NR⁷ wherein R⁷        is selected from hydrogen, hydroxy, C₁₋₄ alkyl and C₁₋₄ alkoxy;    -   R² is selected from hydroxy, halogen, C₁₋₄ alkyl, C₂₋₄ alkenyl,        C₁₋₄ alkoxy, C₂₋₄ alkenyloxy, amino, C₁₋₄ alkylamino, di(C₁₋₄        alkyl)amino; C₁₋₄ alkylthio, C₂₋₄ alkenylthio, nitro, cyano,        —O—CO—R′, —CO—O—R′, —CO—NR′R″, —COR′, —S(O)R′ and —S(O)₂R′,        wherein each R′ and R″ is the same or different and represents        hydrogen, C₁₋₄ alkyl or C₂₋₄ alkenyl; and    -   m is 0 or an integer from 1 to 4;        or a salt thereof, which method comprises mixing powdered carbon        with a compound as defined above for a time sufficient to allow        the compound to partially intercalate within the carbon, and        isolating the resulting modified carbon.

The individual components and aspects of the second preferred aspect ofthe invention will now be described in more detail.

As used herein, a C₁₋₄ alkyl group or moiety is a linear or branchedalkyl group or moiety containing from 1 to 4 carbon atoms. Examples ofC₁₋₄ alkyl groups and moieties include methyl, ethyl, n-propyl,i-propyl, n-butyl, i-butyl and t-butyl. For the avoidance of doubt,where two alkyl moieties are present in a group, the alkyl moieties maybe the same or different.

As used herein, a C₂₋₄ alkenyl group or moiety is a linear or branchedalkenyl group or moiety containing from 2 to 4 carbon atoms. For theavoidance of doubt, where two alkenyl moieties are present in a group,the alkenyl moieties may be the same or different.

As used herein, a halogen is typically chlorine, fluorine, bromine oriodine. It is preferably chlorine, fluorine or bromine.

As used herein the term amino represents a group of formula —NH₂. Theterm C₁₋₄ alkylamino represents a group of formula —NHR′ wherein R′ is aC₁₋₄ alkyl group, preferably a C₁₋₄ alkyl group, as defined previously.The term di(C₁₋₄ alkyl)amino represents a group of formula —NR′R″wherein R′ and R″ are the same or different and represent C₁₋₄ alkylgroups as defined previously. As used herein the term amido represents agroup of formula —C(O)NH₂.

As used herein, an alkoxy group is typically a said alkyl group attachedto an oxygen atom. Similarly, alkenyloxy groups are typically a saidalkenyl group attached to an oxygen atom.

An alkylthio group is typically a said alkyl group attached to a thiogroup. Similarly, alkenylthio groups are typically a said alkenyl groupattached to a thio group.

The alkyl and alkenyl groups or moieties in the compounds used in theinvention are unsubstituted or substituted by one, two or threesubstituents which are the same or different and are selected fromhydroxy, halogen and unsubstituted C₁₋₁₂ alkoxy substituents.

As used herein, the term “partially intercalated” means that thecompound defined above is partially located between sheets of carbon,rather than being located entirely between such sheets. The compound islocalised within edge-plane defect sites along the surface of thecarbon, which can cause a slight increase in the interlayer spacing ofthe carbon. It has been found that the new materials having compoundsdefined above partially intercalated within can be manufactured bysimple processes where the compounds spontaneously partially intercalatewithout the need for long reaction times or coupling agents.

This partial intercalation contrasts with known materials havingintercalating compounds which are fully intercalated. When fullintercalation occurs, the intercalating compounds are located entirelybetween adjacent sheets of graphite and have the effect of pushing thesesheets wide apart. This significantly increases the interlayer spacingof the carbon, which in turn allows subsequent intercalation of other,larger species. Production of carbon having fully intercalated compoundsrequires more experimentally and synthetically complicated processesthan the new methods of the present invention.

Various methods can be used to determine whether a compound definedabove has been partially intercalated within the carbon, as shown by theExamples which follow. A particularly useful technique is X-raydiffraction, which can distinguish between partially intercalatedcompounds and fully intercalated compounds. If the compound is fullyintercalated in the carbon, then when the modified carbon is subjectedto X-ray diffraction a peak will be observed corresponding to aconsiderably larger interlayer spacing than the native carbon which hasnot been modified. However, if the compound is partially intercalated, abroader peak will be observed, with the average interlayer spacing beingthe same as or slightly larger than the usual interlayer spacing for thecarbon used. In particular, when such compounds are partiallyintercalated into multi-walled carbon nanotubes a significantly broaderpeak is observed at a slightly increased interlayer spacing.

Carbon

One form of carbon which is particularly suitable for use in theinvention is graphite. The graphite is preferably in the form ofpowdered graphite. A suitable particle diameter is from 0.1 to 100 μm,more preferably from 1 to 50 μm and more preferably from 2 to 20 μm.

Another form of carbon which is particularly suitable for use in theinvention is multi-walled carbon nanotubes. Carbon nanotubes (CNTs, alsoreferred to herein as nanotubes) have been known for a number of years,having been discovered in 1991 (see S. Iijima, Nature, 1991, 56, 354).One field that has seen a large expansion in the study and use ofnanotubes is electrochemistry. Carbon nanotubes are particularly usefulin this field due to their noted mechanical strength, structure and goodelectrical conductivity. These properties have been used inelectroanalytical applications ranging from catalytic detection andanalysis of biological molecules such as dopamine, cytochrome c andcarbohydrates, to the sensing of analytes such as hydrogen peroxide,hydrazine and TNT.

Structurally, nanotubes approximate to “rolled up” sheets of graphiteand as such are relatively hydrophobic in nature. There are two mainconfigurations of these “rolled up” sheets: single-walled carbonnanotubes (SWCNTs) which are formed as a single, hollow, graphite tube,and multi-walled carbon nanotubes (MWCNTs) which consist of severalconcentric graphite tubes fitted one inside the other. In the presentinvention MWCNTs can be used.

Suitable nanotubes include those purchased from Nanolab Inc. (Brighton,MA, USA). The physical properties of the nanotubes can be optimised bythe person skilled in the art, although exemplary nanotubes have adiameter of from 1 to 50 nm, preferably from 5 to 30 nm, and a length offrom 1 to 50 μm, preferably from 5 to 30 μm. Preferably the carbonnanotubes have a relatively high purity, preferably from 80 to 100%,more preferably from 90 to 100%, most preferably from 95 to 100%.

Partially Intercalating Compounds

The compounds used in the invention are described earlier. The compoundsmay be in the form of nitrobenzene derivatives of formula (I) or saltsthereof. The alkyl and alkenyl groups or moieties in the compounds areunsubstituted or substituted by one, two or three substituents which arethe same or different and are selected from hydroxy, halogen andunsubstituted C₁₋₂ alkoxy substituents.

Preferably the R² substituent is selected from hydroxy, halogen, C₁₋₄alkyl, C₂₋₄ alkenyl, C₁₋₄ alkoxy, C₂₋₄ alkenyloxy, amino, C₁₋₄alkylamino, di(C₁₋₄ alkyl)amino, C₁₋₄ alkylthio, C₂₋₄ alkenylthio, nitroor cyano. More preferably R² is selected from hydroxy, halogen, C₁₋₄alkyl, C₁₋₄ alkoxy, amino, C₁₋₄ alkylamino and di (C₁₋₄ alkyl)amino.More preferably R² is selected from hydroxy, halogen, C₁₋₄ alkyl andC₁₋₄ alkoxy.

The number of R² substituents can vary between zero and four. Preferablythe number of R² substituents, represented by m, is zero, one or two.More preferably m is zero or one, most preferably zero.

X represents a group of formula —(CR⁵R⁶)_(n)— where n is zero or aninteger from one to four.

Preferred R⁵ and R⁶ groups include hydrogen, hydroxy, C₁₋₂ alkyl andC₁₋₂ alkoxy, with hydrogen being preferred.

The alkyl groups or moieties in the X group are unsubstituted orsubstituted by one, two or three substituents which are the same ordifferent and are selected from hydroxy, halogen and unsubstituted C₁₋₂alkoxy substituents. Preferably the R⁵ and R⁶ groups in the X group areunsubstituted.

Preferably n is zero, one or two. Most preferably n is one.

The group Y is selected from hydrogen, hydroxy, C₁₋₄ alkyl and —NR³R⁴wherein R³ and R⁴ are the same or different and are selected fromhydrogen, hydroxy, C₁₋₄ alkyl and C₁₋₄ alkoxy.

The alkyl groups or moieties in Y are unsubstituted or substituted byone, two or three substituents which are the same or different and areselected from hydroxy, halogen and unsubstituted C₁₋₂ alkoxysubstituents. Preferably the alkyl groups or moieties in Y areunsubstituted.

Preferred Y groups are —NR³R⁴. When Y is —NR³R⁴, R³ and R⁴ are the sameor different and are preferably hydrogen, hydroxy, C₁₋₂ alkyl or C₁₋₂alkoxy. More preferably R³ and R⁴ are selected from hydrogen and C₁₋₂alkyl. Most preferably both R³ and R⁴ are both hydrogen.

Preferred compounds are nitrobenzene derivatives of formula (II):

wherein:

-   -   R² is selected from hydroxy, halogen, C₁₋₄ alkyl and C₁₋₄        alkoxy;    -   m is 0, 1 or 2;    -   X represents a group of formula —(CR⁵R⁶)_(n)— wherein n is 0, 1        or 2 and R⁵ and R⁶ are the same or different and are selected        from hydrogen, hydroxy, C₁₋₄ alkyl, C₁₋₄ alkoxy; and    -   Y is selected from hydrogen, hydroxy, C₁₋₄ alkyl and —NR³R⁴        wherein R³ and R⁴ are the same or different and are selected        from hydrogen, hydroxy, C₁₋₄ alkyl and C₁₋₄ alkoxy,        or salts thereof.

Further preferred compounds are nitrobenzene derivatives of formula(III):

wherein:

-   -   R² is selected from hydroxy, halogen, C₁₋₄ alkyl and C₁₋₄        alkoxy;    -   m is 0, 1 or 2;    -   X represents a group of formula —(CR⁵R⁶)_(n)— wherein n is 0, 1        or 2 and R⁵ and R⁶ are the same or different and are selected        from hydrogen, hydroxy, C₁₋₄ alkyl and C₁₋₄ alkoxy; and    -   R³ and R⁴ are the same or different and are selected from        hydrogen, hydroxy, C₁₋₄ alkyl and C₁₋₄ alkoxy,        or salts thereof.

Preferably m is zero or one, more preferably m is zero. Preferably n isone, with R⁵ and R⁶ being the same or different and selected fromhydrogen, hydroxy, C₁₋₄ alkyl and C₁₋₄ alkoxy. More preferably R⁵ and R⁶are the same or different and are selected from hydrogen and C₁₋₄ alkyl.Most preferably both R⁵ and R⁶ are hydrogen.

More preferred compounds (used in the invention) are nitrobenzenederivatives of formula (IV):

wherein R³ and R⁴ are the same or different and are selected fromhydrogen, hydroxy, C₁₋₄ alkyl and C₁₋₄ alkoxy, or salts thereof. Morepreferably R³ and R⁴ are the same or different and are selected fromhydrogen and C₁₋₄ alkyl.

The compounds used to make the compositions of the second preferredaspect of the invention include salts of the nitrobenzene derivatives offormulae (I) to (IV). These salts can be any suitable salt which allowsdoes not interfere with the electrochemical mechanisms which allow thecompounds to be useful in electrodes and electrochemical sensors. Inparticular, there can be mentioned inorganic and organic acid additionsalts. Suitable inorganic acids which can be used include hydrochloric,sulphuric, phosphoric, diphosphoric, hydrobromic or nitric acid.Suitable organic acids which can be used include citric, fumaric,maleic, malic, ascorbic, succinic, tartaric, benzoic, acetic,methanesulphonic, ethanesulphonic, benzenesulphonic orp-toluenesulphonic acid. The salts may also be formed with bases, forexamples with alkali metal (e.g. sodium or potassium) and alkaline earthmetal (e.g. calcium or magnesium) hydroxides and organic bases such asalkyl amines, aralkyl amines or heterocyclic amines.

Without wishing to be bound by theory, it is believed that the compoundsused in the invention partially intercalate into localised edge-planedefect sites along the carbon surface of both graphite and carbonnanotubes. The redox characteristics of carbon modified with thesecompounds show that the modified materials can be used inelectrochemical sensors, for example for the measurement of pH.

In particular, the compounds used in the invention and the modifiedcarbon of the invention are useful in the manufacture of electrodes formeasuring pH. In this regard, the voltammetry of such compounds has beenstudied. The voltammetry of such compounds sensitive to pH, whenimmobilised as molecular solids onto the surface of an electrode, hasbeen found to exhibit Nernstian behaviour which can be describedaccording to the following Nernst equation (1):

$\begin{matrix}{E_{p} = {E_{f}^{0} - {\frac{2.3\mspace{14mu} {RTm}}{nF}{pH}}}} & (1)\end{matrix}$

where E_(p)/V is the peak potential, E⁰ _(f)/V is the formal potentialof the redox couple, R/J K⁻¹ is the universal gas constant, T/K is thetemperature and m and n are the number of protons and electrons involvedin the redox process respectively.

Accordingly, by studying the voltammetric response of these compounds,for example using cyclic voltammetry or square-wave voltammetry, alinear response of peak potential to pH would be expected.

Other Redox Active Materials

As well as the compounds defined above, other redox active materials canbe included in the materials of the invention. These additional redoxactive materials may be any organic material capable of undergoingelectron loss and gain. Preferably the additional redox active materialis a solid phase material. When immobilised onto a substrate, e.g.glassy carbon or a basal plane pyrolytic graphite (bppg) electrode, itundergoes concomitant proton and electron loss/gain onoxidation/reduction.

The additional redox active material may be sensitive or insensitive tothe species which is to be detected or measured. In either case, bymeasuring the potential difference between the current peaks for thecompounds defined earlier and for the additional redox active material,the concentration of the species to be measured can be determined.

It is preferred that the electrodes of the invention be useful in themanufacture of pH meters, and accordingly in one embodiment theadditional redox active material is sensitive to the concentration ofprotons. Preferably the peak potential of the additional redox activematerial depends on the local proton concentration. As discussed abovein relation to the compounds used in the invention (i.e. thenitrobenzene derivatives or salts thereof) the voltammetry of redoxactive materials which are sensitive to pH has been found to showNernstian behaviour.

Accordingly, by studying the voltammetric response of these compounds,for example using cyclic voltammetry or square-wave voltammetry, alinear response of peak potential to pH would be expected.

More than one additional redox active material may be used in theinvention. Suitable additional redox active materials include quinonesand anthracenes, for example 9,10-anthracene, 9-nitroanthracene,phenanthraquinone (PAQ) and 1,2-napthaquinone (NQ). Other materials thatcan be used include azobenzene, diphenylamine, methylene blue,3-nitrofluoranthene, 6-nitrochrysene and thionin.

When present, the additional redox active material can be combined withthe modified carbon by any suitable process. For examples, in oneembodiment of the invention the additional redox active material can becombined with the modified carbon by chemisorption of aryldiazoniumsalts using hypophosphorous acid as the chemical reducing agent. Inanother embodiment of the invention phenanthraquinone (PAQ) can bephysisorbed onto graphite.

In another embodiment of the invention the additional redox activematerial can be combined with the modified carbon described previouslyby way of agglomeration. Such agglomerates comprises (i) carbonnanotubes having a compound as defined above which is a nitrobenzenederivative of formula (I) or a salt thereof partially intercalatedwithin, and (ii) a binder, wherein the binder is the additional redoxactive material. The nanotubes, compound and additional redox activematerial may be as described above.

In this embodiment, the agglomerate is made by dispersing the nanotubesin a binder. The preferred method comprises combining MWCNTs having acompound of as defined above partially intercalated within (hereafter“the modified MWCNTs”) and binder material in a solvent, and thenprecipitating the agglomerate out of the solution. In particular, themethod may comprise:

-   -   (1) combining the modified MWCNTs and the binder in a solvent;    -   (2) adding an excess of aqueous solution in order to cause        precipitation of the agglomerate out of the solvent; and    -   (3) recovering the agglomerate.

Preferably the solvent is a hydrophobic solvent, comprising smallorganic molecules. The solvent should be chosen such that the redoxactive compound and the carbon nanotubes are both soluble within it.Suitable solvents include all common organic solvents such as acetone,acetonitrile and dimethyl formamide.

In this embodiment, it is preferred that the additional redox activematerials are hydrophobic, having a low solubility in water. This allowsthem, when an agglomerate is being manufactured, to mix with the carbonnanotubes in solution and results in the agglomerate precipitating outof solution when an excess of aqueous solution is added.

The agglomerate preferably comprises the modified MWCNTs and additionalredox active materials only, with no other materials present. However,the agglomerate may contain some impurities such as residual solvent,left as a result of a process by which the agglomerate is be produced.Preferably these impurities comprise less than 1 wt % of theagglomerate, more preferably less then 0.5 wt %. The precise level ofimpurities which is acceptable in the agglomerate will depend upon howthe impurities affect the voltammetry of the agglomerate.

The size of the agglomerates depends upon the nature and proportions ofthe components used in their preparation and the conditions of theprocess by which they are prepared. However, exemplary agglomerates maybe approximately 10 μm in diameter and consist of bundles of nanotubesrunning into and throughout an amorphous molecular solid which binds theagglomerate together.

The agglomerate is preferably applied to the substrate of the electrodeby way of abrasive immobilisation.

The Substrate

The substrate onto which is applied the modified carbon may be anysubstrate conventionally used in the manufacture of electrodes. Forexample, the substrate may be a basal plane pyrolytic graphite (bppg)electrode or glassy carbon, metal electrodes such as gold or platinum,or optically transparent electrodes such as those comprising ITO. Thesubstrate preferably has good electrical contact with the carbonnanotubes, and also has a surface such that good coverage with thecarbon nanotubes and redox active material can be achieved.

The Sensor

The structure of sensors according to the invention will depend upon thefinal application of the sensor, and depends upon the substance whichthe sensor is to measure and the environment in which measurement willtake place. Known sensor structures may be employed in conjunction withthe agglomerates and electrodes described herein.

Exemplary sensors may have a two or three terminal arrangement. Thus,they may comprise a working electrode of the invention and a combinedcounter and reference electrode, or a working electrode, counterelectrode and separate reference electrode. The reference electrode andcounter electrode can be any conventional electrodes known in the art.

The materials used in the sensor depend upon which species the sensor isintended to measure and the environment in which the sensor is to beused. In order to modify the sensor to be sensitive to a differentspecies it is simply required for the skilled person to substitute thepartially intercalating compound defined above (i.e. the nitrobenzenederivative or salt thereof) or additional redox active material with adifferent partially intercalating compound or additional redox activematerial sensitive to the species which is to be measured.

The modified carbon materials of the invention are particularly suitedfor use in “single-shot” pH sensors for use in “dirty” environments,such as effluent or sewage, where recovery of the sensor is likely to beundesirable.

Method for Preparing the Modified Carbon

Carbon is modified according to the method of the invention by mixingthe carbon in a solvent with a partially intercalating compound definedabove. Suitable solvents include common aprotic organic solvents. Forexample, there can be mentioned dimethyl formamide (DMF),tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), acetone, acetonitrile,ethyl acetate, chloroform, methylene chloride. The solvent may bedegassed or may contain dissolved gases, e.g. dissolved oxygen.

The carbon, solvent and compound are mixed for a time sufficient toallow partial intercalation of the compound into the carbon. A suitabletime is 1 to 5 hours, although longer or shorter time periods can alsobe used. After this time the modified carbon is filtered, washed withsolvent in order to remove any physisorbed species, and dried. Oneparticular advantage of this method is that it can proceed without theneed for a coupling agent.

The relative amount of the compound and carbon can be chosen by theskilled person according to the end use of the modified carbon.

Surprisingly the compounds used in the invention spontaneously partiallyintercalate into the carbon. The resulting materials are robust, forexample when used in electrochemical sensors, producing stable responsesat elevated temperatures. Furthermore, the compounds partiallyintercalate at a high level, i.e. a large amount of the compounds can bepartially intercalated. This results in large current response and highsensitivity.

Examples of the Second Preferred Aspect of the Invention

All reagents were obtained from Aldrich (Gillingham, UK) with theexception of potassium chloride (Riedel de Haën, Seelze, Germany)tetrabutylammonium perchlorate (TBAP), lithium perchlorate (FlukaChemicals, Gillingham, UK) acetonitrile (synthesis grade, 99.99%anhydrous, Fischer Scientific, Loughborough, UK) diethyl ether (BritishDrug House Chemicals, Poole, UK) and were of the highest grade availableand used without further purification.

4-nitrobenzylamine (4-NBA) was obtained as the hydrochloride salt. Inorder to liberate the free amine the following procedure was used:4-nitrobenzylamine hydrochloride salt (2.0 g, 0.011 mmol) was dissolvedin water (40 cm³) and sodium hydroxide (20 cm³ of a 1M aqueous solution)was added. The solution was stirred for 2 hours, after which time thesolution was washed with diethyl ether (2×50 cm³). The combined organiclayers were washed with brine (50 cm³), dried over MgSO₄, filtered andconcentrated in vacuo to afford 4-nitrobenzylamine (1.2 g, 78% yield) asa red crystalline solid which was used without further purification. Thecorresponding nuclear magnetic resonance spectrum was recorded andcompared with library spectra in order to confirm that the pure compoundhad been re-crystallised. The crystals of 4-NBA were stored in anair-tight container at 4° C. prior to use.

Aqueous solutions were prepared using deionised water from an Elgastat(Elga, UK) UHQ grade water system with a resistivity of not less than18.2 MO cm. Non-aqueous solutions were prepared using acetonitrile(supplied as 99.99% anhydrous), which was dried over 5 A molecularsieves for 24 hours prior to use to remove any trace water content.Cyclic voltammetric measurements were made after degassing the solutionwith pure N₂ gas (BOC gases, Guildford, Surrey, UK) for 30 minutes andcarried out at 20±2° C.

Synthetic graphite powder (2-20 μm diameter) was purchased from Aldrich.Multiwalled carbon nanotubes (purity >95%, diameter 10-40 nm, length5-20 μm) were purchased from NanoLab Inc. (Brighton, MA, USA) and wereused without further purification.

Solutions of known pH in the range pH 1.0 to pH 12.0 were made up indeionised water as follows: pH 1.0, 0.10 M HCl; pH 4.6, 0.10 M aceticacid+0.10 M sodium acetate; pH 6.8, 0.025 M Na₂HPO₄+0.025 M KH₂PO₄; pH9.2, 0.05 M disodium tetraborate; pH 12, 0.01M sodium hydroxide. Thesesolutions contained in addition 0.10 M KCl as supporting electrolyte. pHmeasurements were performed using a Jenway 3030 pH meter.

Electrochemical measurements were recorded using a μAutolab computercontrolled potentiostat (Ecochemie, Utrecht, Netherlands) with astandard three-electrode configuration. All experiments were carried outin a glass cell of volume 25 cm³. A basal plane pyrolitic graphiteelectrode (bppg, 0.20 cm², Le Carbone Ltd, Sussex, UK) acted as theworking electrode (see below). A platinum coil (99.99% Goodfellow,Cambridge, UK) acted as the counter electrode. The cell assembly wascompleted either by using a saturated calomel electrode (SCE,Radiometer, Copenhagen, Denmark) as a reference electrode in aqueoussolution, or by using a silver wire (99.99% Goodfellow, Cambridge, UK)as a quasi-reference electrode in non-aqueous solution.

Unless stated otherwise cyclic voltammograms were recorded using thefollowing parameters: step potential 2 mV, scan rate 100 mVs⁻¹.Square-wave voltammetric parameters were as follows: frequency 12.5 Hz,step potential 2 mV and amplitude 25 mV.

Scanning electron microscopy (SEM) images were recorded using a Jeol6500F instrument. High resolution transmission electron microscopy(HRTEM) images were recorded using a Jeol 2010F instrument.

X-ray powder diffraction experiments were carried out using aPanalytical Xpert Pro instrument utilising X-ray radiation from thecopper Kα₁ band (λ=1.54 Å).

Example 5 Protocol for the Derivatisation of Graphite Powder andMultiwalled Carbon Nanotubes (MWCNTs) with 4-NBA and their AbrasiveImmobilisation onto the Surface of a Bppg Electrode

Derivatisation of graphite powder (0.5 g) or MWCNTs (50 mg) with4-nitrobenzylamine (4-NBA) was achieved by stirring either the graphitepowder sample or the MWCNT sample in a solution of 4-NBA in acetonitrile(10 mM, 25 cm³) for 120 minutes at room temperature unless otherwisestated. Next, the sample was filtered under suction, washed withacetonitrile (5×50 cm³) to remove any physisorbed species and air driedfor 12 hours. After which, the sample was stored in an air-tightcontainer prior to use.

The derivatised carbon powders or MWCNTs were then abrasivelyimmobilised onto the surface of a clean bppg electrode prior to anyelectrochemical experiment. This was achieved by initially polishing thebppg electrode on glass-polishing paper (H00/240), after which it waspolished on silicon carbide paper (P1000C) for smoothness. The4-NBAcarbon (i.e. graphite having 4-NBA partially intercalated within)or 4-NBAMWCNTs (i.e. MWCNTs having 4-NBA partially intercalated within)were then abrasively immobilised onto the bppg electrode by gentlyrubbing the electrode surface on a fine filter paper (Whatman)containing the 4-NBAcarbon or 4-NBAMWCNTs.

Example 6 Voltammetric Characterisation of Graphite Powder and MWCNTsDerivatised with 4-Nitrobenzylamine

Cyclic voltammetry (CV) was used in order to confirm that 4-NBAmolecules are attached onto the graphite powder (4-NBAcarbon), or ontothe MWCNTs (4-NBAMWCNTs), which themselves are abrasively immobilisedonto a bppg electrode. A standard protocol (given below) was employedover the entire pH range (pH 1.0 to pH 12.0).

First, twenty repeat scans were recorded to ensure that the redoxspecies in question (4-NBA) is stable when in contact with an aqueoussolution and does not desorb from the electrode surface. FIG. 15A showstwenty repetitive cycles of 4-NBAcarbon on bppg in pH 6.8 buffer. As canbe seen from FIG. 3A, it is necessary to sweep the potential in areducing direction to ca. −1.0 V vs. SCE, past an irreversible redoxprocess at ca. −0.6 V vs. SCE (labelled as “system I” in FIG. 15A andFIG. 14), in order to generate an electrochemically almost-reversiblecouple at ca −0.1 V vs. SCE (labelled as “system II” in FIG. 15A andFIG. 14). The exact potentials of the redox processes discussed abovewill depend on the pH of the solution. At every pH studied in the rangepH 1.0 to pH 12.0 a wave shape that was almost symmetrical and had aslight peak to peak separation that increased with increasing scan ratewas observed. It was found that after twenty repetitive scans the peakcurrents (which were initially found to decrease slightly) remainedstable and that the charges (peak areas) of both the oxidative andreductive peak processes were almost equal to each other (FIG. 15A).

The next step was to replace the electrolyte solution with freshsolution and record the voltammetric response. The corresponding cyclicvoltammograms were found to overlay the last scan at every pH studied,thereby confirming that the electroactive species remains on theelectrode and is not released to solution. FIG. 15B shows the overlaidCVs after replacing pH 6.8 solution with fresh solution.

Finally, after formation of the reversible couple (system II), asdescribed above, the scan rate was varied from 25 to 900 mVs⁻¹ (FIG.15C) and a plot of peak current versus scan rate (FIG. 15D) was found tobe almost linear, consistent with a surface bound species. However thepeak separation of(ca. 100 mV at low scan rates) is considerably largerthan the theoretical zero peak to peak separation for an ideal,immobilised, electrochemically reversible species. These discrepanciesmay possibly be due to some slight ohmic distortion at higher scan ratesand/or electrode kinetic factors. In fact the wave shapes and thevariation of peak potential with increasing scan rate suggest that anelectrochemically quasi-reversible immobilised system exists over theentire pH range studied.

Example 7 Voltammetric Response of 4-NBAcarbon and 4-NBAMWCNTs from pH1.0 to pH 12.0

Having characterised these materials from pH 1.0 to pH 12.0 using cyclicvoltammetry, this Example now demonstrates that these modified carbonmaterials can be used for the analytical sensing of pH. As shown in FIG.14, the electrochemical reduction of aromatic nitro compounds such as4-NBA undergoes an initial irreversible step involving four-electronsand four-protons to form the corresponding arylhydroxylamine. Uponrepetitive cycling the aryihydroxylamine can then undergo a chemicallyreversible, electrochemically quasi-reversible oxidation to thecorresponding aryl nitroso compound, involving a further two-electronsand two-protons.

The peak potentials for these processes must therefore depend on thelocal proton concentration and hence must be sensitive to variations inpH. The variation in peak potential with pH can be described by theNernst equation (1) discussed earlier. In this Example n=m and is likelyto be equal to two in the case of the reversible process (system II inFIG. 14).

FIG. 16A shows the overlaid cyclic voltammograms (first scan) recordedin a range of solutions of differing pH (pH 1.0, 0.10 M HCl; pH 4.6,0.10 M acetic acid+0.10 M sodium acetate; pH 6.8, 0.025 M Na₂HPO₄+0.025M KH₂PO₄; pH 9.2, 0.05 M disodium tetraborate; pH 12.0, 0.01M sodiumhydroxide) for 4-NBAcarbon on bppg. A plot of peak potential vs. pH forthe peak corresponding to system I in FIG. 14 and both the oxidative andreductive peaks corresponding to system II in FIG. 2 is presented inFIG. 16B. The plots of peak potential vs. pH produced in each case alinear response with a corresponding R² value of not less than 0.9943.The gradient of such a plot for the oxidative and reductive peaks ofsystem II yielded values of 60.4 mV/pH unit and 59.8 mV/pH unitrespectively. This is close to the ideal theoretical gradient of 59.1mV/pH unit at 298 K predicted by the Nernst equation.

A similar plot of peak potential vs. pH for the irreversible system Iyielded a gradient of 42.5 mV/pH unit. This clearly deviates from theNernstian behaviour predicted by the equation (1). This may be partlyexplained by the fact that the Nernst equation (1) is derived forsystems exhibiting reversible kinetics, 1 whilst system I is clearlychemically and electrochemically irreversible. Furthermore similarresults of a ca. 40 mV/pH unit shift were obtained by Wain et al. intheir studies of microdroplets of 4-nitrophenol-nonyl-ether on a bppgelectrode immersed in aqueous solutions of varying pH. They attributedthis deviation from Nernstian behaviour to possible competition betweenH⁺ and alkali metal cations such as K⁺ and especially Li⁺. The fact thatsystem I rapidly vanishes after the first couple of scans makes thesemodified carbon materials of the invention ideally suited for use as“single-shot” pH sensors for use in media, such as effluent andsewerage, where recovery of the sensor is unlikely to be desirable.

The reversible, Nernstian redox processes corresponding to theoxidation/reduction of the arylhydroxylamine/arylnitroso moieties of4-NBA remain stable for many tens of scans. The variation of this redoxcouple with pH was investigated further using the technique of squarewave voltammetry. Square wave voltammetry has significant advantagesover conventional cyclic voltammetry, as it provides a means of carryingout a single sweep, producing a well-defined voltammetric peak as thearylhydroxylamine/arylnitroso couple has nearly reversible kineticbehaviour. FIG. 17A shows the corresponding overlaid reductive squarewave voltammograms recorded at each pH studied for 4-NBAMWCNTs. It isworth noting that at pH 1.0 the oxidative wave is somewhat distorted dueto the presence of an azoxy linkage affecting the redox chemistry. Thiscauses some deviation from linearity in a plot of peak potential vs. pHat low pH values (FIG. 17B). Furthermore an extra peak is observed at pH12.0 that was not observed in the cyclic voltammograms at this pH. Thepresence of this peak can be attributed to the formation of the radicalanion of the nitro-group upon reduction which is relatively stable andelectrochemically reversible at such alkaline pHs. Its presence isobserved in square wave but not in cyclic voltammetry due to the highsensitivity of the technique. It is apparent from FIG. 17B that a plotof peak potential vs. pH taken from the square wave data produces alinear, Nernstian response (apart from the deviation at low pH), withgradients of 62.8 mV/pH unit and 59.2 mV/pH unit for the oxidative andreductive processes respectively and R² values greater than 0.9998. Thisagain is in excellent agreement with theory (equation 1).

Example 8 Characterisation of 4-NBAcarbon and 4-NBAMWCNTs

The nature of the surface modification of the carbon used in theExamples above was then investigated to determine whether the compoundof formula (I) (in this case 4-NBA) had indeed been intercalated and inparticular to determine whether it had been partially intercalated. Interms of modification of carbon by a compound, there are three generalpossibilities: (i) physical adsorption (physisorption), (ii) chemicaladsorption (chemisorption) and (iii) full or partial intercalation.

Example 8.1 Test for Physical Adsorption (Physisorption) of 4-NBA ontoGraphite and MWCNTs

The inventors investigated how the length of time the reaction mixturewas stirred during the derivatisation procedure affected the amount of4-NBA absorbed by the carbon material. In order to do this an aliquot ofthe reaction mixture, which contained graphite particles or MWCNTssuspended in a 10 mM solution of 4-NBA in acetonitrile, was removed,filtered, washed with dry acetonitrile and dried at 40 minute intervalsuntil 160 minutes of stirring had elapsed. The samples were thenabrasively immobilised onto a bppg electrode and cyclic voltammetry wascarried out in pH 6.8 buffer. Five scans were recorded for each sample,and five samples were separately abrasively immobilised for each aliquotremoved at a given time. From the five cyclic voltammograms recorded foreach sample, the peak areas corresponding to the irreversiblefour-electron, four-proton reduction of the nitro-group moiety at −0.6 Vvs. SCE in FIG. 15A, and the reversible two-electron, two-protonarylhydroxylamine/arylnitroso couple at −0.1 V vs. SCE in FIG. 3A weremeasured. These peaks are labelled as system I and system IIrespectively in FIG. 14.

The peak area is the amount of charge passed during a redox process andcan therefore be directly related to the number of moles of 4-NBA on thecarbon surface using Faraday's Laws. This was repeated for each of thefive samples taken at each time interval. Because the exact amount ofmaterial immobilised onto the electrode surface cannot be accuratelycontrolled using abrasive immobilisation, the data from each of the fivecyclic voltammograms recorded for each of the five samples (twenty-fivecyclic voltammograms in total for each time period investigated) wasaveraged and plotted against time. The standard deviation was calculatedas an error bar for each point showing the dispersion of data over thefive abrasive immobilisations carried out for each time interval.

FIG. 18 shows a plot of peak area vs. time recorded for the irreversiblereduction peak at −0.6 V vs. SCE of 4-NBA derivatised graphite. Alsoshown on this graph for comparison are the theoretical maximum chargespassed for a monolayer of 4-NBA molecules covering the geometric area ofthe bppg electrode, and for a monolayer of 4-NBA coating the surface ofthe graphite particles (modelled as spheres) which themselves areclose-packed as a monolayer on the geometric surface of the electrode.This model is a gross oversimplification as first the true surface areaof a bppg electrode is always larger than its geometric area due to thesurface not being perfectly smooth, and second the abrasiveimmobilisation of graphite particles onto the electrode surface islikely to produce multiple layers of particles which are not necessarilygoing to be close packed due to the uneven shape and size distributionsof the particles. However such a calculation is useful in that itprovides us with an indication of the degree of modification by the4-NBA molecules.

FIG. 18 shows that at longer reaction times the amount of immobilised4-NBA reaches a maximum value showing saturation. Analogous results wereobserved for both the oxidative and reductive peaks belonging to systemII in FIG. 2, and in the case of MWCNT derivatisation. Whilst thisexperiment in itself does not provide evidence for intercalation itgives an interesting insight into the “filling-up” of the sites at which4-NBA can modify the carbon surface. This effect would also be observedif physisorption led to only a monolayer formed on the surface. In factit is important to note that the fact that the number of sites where4-NBA can modify the MWCNTs or graphite powder is limited is consistentwith any of the three hypothesis presented in this report, physicaladsorption, chemical adsorption or intercalation of 4-NBA.

The derivatised carbon powders/MWCNTs are washed with a large quantityof dry acetonitrile during the derivatisation procedure, with thespecific aim of removing any physisorbed material which is known todesorb when treated with non-aqueous solvents. It is therefore highlyimprobable that surface physisorption is the mechanism of modificationin this case.

Further evidence against physisorption arises from the fact that we havecarried out electrochemical experiments using cyclic voltammetry on4-NBAcarbon and 4-NBAMWCNTs in acetonitrile solutions with 0.1M TBAP and0.1M LiClO₄ as supporting electrolyte (see below). However, slowdesorption kinetics of 4-NBA from the MWCNTs or graphite powder mayexplain the fact that voltammetry can be observed from 4-NBAcarbon and4-NBAMWCNTs in acetonitrile. Therefore in order to verify that slowdesorption kinetics were not responsible for our experimentalobservations described above, the following experiment was performed. Inthis experiment, 4-NBAcarbon was abrasively immobilised onto a bppgelectrode. The electrode was then immersed into acetonitrile and storedfor a period of one week. After this time the electrode was removed andplaced in an aqueous solution and the cyclic voltammetry recorded. Nosignificant deterioration in the aqueous voltammetric response, eitherin the magnitude of peak currents or peak potentials was observed.

Next cyclic voltammetry was conducted on immobilised 4-NBA inacetonitrile solutions containing either 0.1M TBAP or 0.1M LIClO₄ assupporting electrolyte. Upon sweeping in a reductive directionvoltammetric peaks were observed with each electrolyte saltcorresponding to the reduction of the attached nitro-group moiety. With0.1M TBAP as supporting electrolyte a poorly resolved electrochemicallyreversible couple was observed at ca.−1.0 V vs. Ag. This can beattributed to the reversible one-electron reduction of the nitro-groupto the corresponding radical anion. When 0.1M LiClO₄ was used assupporting electrolyte an irreversible reduction wave was observed atca.−0.8 V vs. Ag and a reversible system was formed on repetitive cyclesat ca.−0.4 V vs. Ag.

Further cyclic voltammetric experiments were carried out using4-NBAcarbon abrasively immobilised onto a bppg electrode in acetonitrilecontaining 0.1M TBAP as supporting electrolyte. The electrode was thenremoved from the non-aqueous solution and placed in an aqueous solution(pH 6.8). Again a stable voltammetric response was observedcorresponding to the aqueous redox electrochemistry of 4-NBA. Next anelectrode containing abrasively immobilised 4-NBAcarbon is placeddirectly into the aqueous electrolyte (pH 6.8) and the voltammetryrecorded. A comparison of the resulting cyclic voltammograms for bothcases (with and without conducting cyclic voltammetry in non-aqueoussolution prior to conducting voltammetry in pH 6.8 buffer) revealed thatthe magnitude of the peak currents and the peak potentials observed inthe voltammetry were almost identical.

If the 4-NBA was physically adsorbed onto the graphite surface one wouldexpect it to have desorbed into the acetonitrile solution, and noresponse would be observed either in the non-aqueous acetonitrilesolution or in the aqueous electrolyte solution.

It can therefore be concluded that physical adsorption is not themechanism by which the carbon surface of graphite or MWCNTs is modifiedwith 4-NBA.

Example 8.2 Test for Chemical Adsorption (Chemisorption) of 4-NBA ontoGraphite and MWCNTs

In previous studies chemisorption of organic molecules onto carbon isachieved in two ways. The first is an “heterogeneous” method which usesthe direct electrochemical reduction of diazonium salts or theelectrochemical oxidation of amines. The second method is carried out“homogeneously” (the term “homogeneous” is used in this context to meanthat both the modifier and the oxidant/reductant are in the solutionphase) e.g. the reduction of aryldiazonium salts with hypophosphorousacid in the presence of graphite powder.

Barbier et al. have shown that 4-NBA can be “heterogeneously” chemicallybound to the surface of a glassy carbon electrode by directelectrochemical oxidation. The mechanism of the amine bond formation tothe electrode surface proceeds via the oxidation of the amine group tothe corresponding radical cation, ArNH₂ ^(+•) which subsequently canreact with the carbon surface to form a covalent C—N bond.

To test whether we had achieved the chemisorption of 4-NBA using a“homogeneous” method the following experiment was carried out: graphitepowder and MWCNTs were derivatised with 4-NBA (10 mM in acetonitrile).Next the derivatisation procedure was repeated on fresh batches ofgraphite and MWCNTs with the exception that the acetonitrile wasdegassed with nitrogen for 20 minutes prior to use. The reaction mixturewas kept under a blanket of nitrogen during the derivatisation procedureto prevent atmospheric oxygen diffusing into the solution. Thisprevented the possibility of aerial oxidation of 4-NBA to the radicalcation by dissolved or atmospheric oxygen and subsequent reaction withthe carbon material. Finally fresh batches of graphite and MWCNTs werederivatised as describe previously except that a strong oxidising agent,(tris(4-bromophenyl)aminium hexachloroantimonate (TBPAHCA, 25 mM inacetonitrile) was added to the reaction mixture. TBPAHCA was used topromote oxidation of the amine group to the corresponding radical cationand subsequent reaction with the carbon material.

The resulting 4-NBA derivatised graphite and MWCNTs from each of thethree different preparations (using a strong oxidant, using acetonitrilewhich contained dissolved oxygen and using degassed acetonitrile) wereseparately immobilised onto a bppg electrode and their correspondingcyclic voltammograms recorded in pH 6.8 buffer and in acetonitrile (0.1MTBAP). In all three cases there was no observable difference in thevoltammetric behaviour of the 4-NBAcarbon or 4-NBAMWCNTs in eitheraqueous or non-aqueous media. It can be inferred from this result thatgraphite and MWCNTs are derivatised by 4-NBA in exactly the same mannerwhether the derivatisation is carried out in degassed acetonitrile,acetonitrile containing dissolved oxygen or in the presence of a strongoxidising agent. This implies that 4-NBA modifies the carbon materialseven when the formation of the corresponding radical cation of the aminegroup is unlikely to occur. Thus it can be concluded that chemicaladsorption of 4-NBA via formation of the radical cation and subsequentattack of the carbon surface is not the likely mechanism of carbonmodification.

One further possible mechanism by which the 4-NBA molecules couldchemically attach themselves to the surface is by reacting with surfacecarboxylic acid groups, which are formed on the surface of syntheticgraphite and MWCNTs during manufacture, to form the correspondingamides. This however is not plausible for two reasons. First, amides aresusceptible to cleavage by hydrolysis at high and low pH, whilst we havedemonstrated by using cyclic voltammetry and observing a stablevoltammetric response over many repeat cycles that 4-NBA remains stableon the carbon surface at pH 1.0 and pH 12.0. Second, experiments havebeen carried out which demonstrate that significant amounts of 4-NBAhave modified the carbon surface stirring in the reaction mixture forjust 40 minutes. In order for amidification to take place on time scalesshorter than several weeks it is customary to use a coupling agent suchas dicyclohexylcarbodiimide (DCC) as a catalyst to facilitatenucleophilic attack by the amine onto the carboxylic acid and to assistin the departure of the OH⁻ leaving group. In the present derivatisationprocedure there is neither a coupling agent nor sufficient time foramidification to occur.

Example 8.3 Test for Full or Partial Intercalation of 4-NBA intoGraphite and MWCNTs: Voltammetric Evidence

Having established in the previous two sections of this Example that4-NBA is unlikely to adsorb onto the surface of graphite powders orMWCNTs the inventors then considered the possibility of full or partialintercalation of 4-NBA at localised edge-plane-like defects on thecarbon surface. First the electrochemical evidence in light of theextensive literature devoted to the subject of graphite intercalationcompounds was studied.

The fact that voltammetry can be observed in non-aqueous solutions, evenafter soaking a 4-NBA modified electrode in acetonitrile for one week,and yet we have shown that 4-NBA is unlikely to have been physisorbed orchemisorbed onto the carbon surface, implies that there may be somepartial intercalation of 4-NBA into the carbon material. This is likelyto occur at edge plane or edge-plane-like defects on the surface ofeither graphite or MWCNTs. Further electrochemical evidence for thisstems from the effect of the supporting electrolyte used in theacetonitrile solutions.

If tetrabutylammonium perchlorate (TBAP) is used as the supportingelectrolyte salt then any features in the cyclic voltammetry of4-NBAcarbon or 4-NBAMWCNTs are small and poorly defined. However if theTBAP salt is replaced with lithium perchlorate then well-definedvoltammetry is observed (FIG. 19). In particular it is worth noting that4-NBA appears to remain bound to carbon in acetonitrile and does notleach into the solution phase, unless it is electrochemically reduced inthe presence of Li⁺ ions. When this occurs, the reduction, unlike in thecase of TBAP, produces well defined peaks in the voltammetry. Aninvestigation of the electrochemically reversible couple observed atcirca −0.4 V vs. Ag reveals that upon repetitive cycles thecorresponding peaks rapidly decrease and have completely disappeared bythe tenth scan. If the experiment is repeated and the solution isagitated between recording the first and second scan by gently stirringthe solution while the electrode is still immersed in it, the peaksassociated with this redox process are no longer present in the secondscan. This implies that the redox species, which is a reduced form of4-NBA is in the solution phase and that stirring of the solution removesthis species from the diffusion layer extending from the electrodesurface into the bulk solution. Hence the signal corresponding to thisspecies is no longer observed in the voltammetry after stirring.

This behaviour can be explained if 4-NBA is partially intercalated atedge-plane defect sites along the surface of the carbon. The NBu₄ ⁺cation may be sterically hindered from approaching and complexing withthe 4-NBA molecule as it lies inside the “pocket” formed by its partialintercalation into the disordered defect site (FIG. 20). Furthermore,the NBu₄ ⁺ cation typically forms weakly bound ion-pair complexes. Hencethe voltammetry of 4-NBAcarbon or 4-NBAMWCNTs observed in acetonitrilecontaining 0.1M TBAP is poorly defined. Lithium cations on the otherhand are much smaller in size and might therefore approach the 4-NBAeven when it is partially intercalated. Upon reduction of thenitro-group the lithium ions, being highly polarizing, can form acomplex with the reduced form of 4-NBA as either the radical anion orthe arylnitroso/arylhydroxylamine. This complexation with Li⁺ ions maycause the [Li 4-NBA]ion pair to leach out into solution. The formationof [Li 4-NBA]ion pairs is supported by the fact that the peak potentialfor the reduction in 0.1M LiClO₄ is shifted in a positive directioncompared to the peak potential in 0.1M TBAP.

This positive shift in peak potential upon complexation with Li⁺ hasbeen well documented in the literature. Initially the [Li 4-NBA]ion pairremains in the diffusion layer adjacent to the electrode surface and sofurther redox voltammetry can be observed. However, upon solutionagitation these ion pairs can be transported into bulk solution outsidethe diffusion layer and thus the corresponding voltammetry is no longerobserved.

The small size of the solvated Li⁴ ion is probably crucial to thisprocess as even when the size of the quaternary ammonium cation isreduced by repeating the experiments with tetraethyl ammonium andtetramethyl ammonium perchlorates (TEAP and TMAP respectively) thevoltammetry remains poorly-defined.

Full intercalation of 4-NBA deep within the inter-layer region in native(i.e. unmodified or treated) graphite or MWCNTs (which are analogous to“rolled-up” sheets of graphite) is highly unlikely. The inter-layerspacing (I_(c)) between ordered graphite sheets is 3.35 Å (I_(c)=3.44 Åin MWCNTs) which is too small to reasonably accommodate a 4-NBA molecule(FIG. 9). In order for full intercalation to occur the inter-layerspacing must increase. An extensive literature search reveals that: (1)there is no evidence of acetonitrile spontaneously intercalating intonative graphite, thus there is no evidence for swelling of the graphitepowder or MWCNTs by immersion into acetonitrile which would facilitate4-NBA intercalation; (2) there is no evidence to suggest organicaromatic molecules similar in size to 4-NBA spontaneously intercalateinto native graphite; (3) in the case of compounds of formula (I) whereY is an amine group, there is no direct evidence for amine intercalation(including ammonia and methylamine) into native graphite. However, allthe above scenarios are possible when the inter-layer spacing (I_(c))between graphite sheets is increased. This is readily achievable byintercalating alkali metal ions such as Li⁺, K⁺ into graphite to producegraphite intercalation compounds (GICs) e.g. C₈Li, C₂₄Li, or by usinggraphitic oxide, and graphitic acid. There is even evidence to suggestthat SWCNTs can intercalate K⁺ and FeCl₃ without rupturing the tubestructure. Hence, the present derivatisation procedure does not includeany of the above systems or criteria, and it can be concluded that fullintercalation of 4-NBA into graphite does not occur.

Example 8.4 Test for Full or Partial Intercalation of 4-NBA intoGraphite and MWCNTs: Evidence from Electron Microscopy and X-Ray PowderDiffraction

Having inferred that intercalation may be responsible for themodification of graphite and MWCNT by 4-NBA using electrochemical means,evidence from other techniques is now presented.

Intercalation is only likely to occur at the edge-planes of graphite.These edge-plane surface sites are numerous on graphite powder particlesand are the site of much of the chemical and electrochemical surfaceactivity. Large regions of these edge-plane defects lead to areas of“disordered” graphite where the well-defined graphite crystal structurebreaks down. Due to the relatively large dimensions of these disordereddomains in graphite powder and the irregular morphology and sizedistribution of such particles (2-20 μm diameter) imaging of the surfacebefore and after modification with 4-NBA using the techniques ofelectron microscopy does not give an insight into the nature and effectof the surface modification. However, MWCNTs have a relatively welldefined size and morphology, and as such, any difference or disparityarising from modifying the surface with 4-NBA should become apparent.

Intercalation of 4-NBA into MWCNTs should cause some degree of expansionin the size of the spacing between adjacent graphite sheets or regionsof graphitic material on the surface causing the tubes to “swell”. Inextreme cases these distortions could even cause the tubes to deformand/or rupture. In order to determine whether any swelling could beobserved in the 4-NBA modified MWCNTs SEM was employed to image bothunmodified “native” MWCNTs and 4-NBA modified MWCNTs after abrasiveimmobilisation onto the surface of a bppg electrode (FIG. 10A and FIG.10B). Careful analysis of the diameter of the CNTs reveals that theaverage diameter of the native, unmodified MWCNTs is ca. 40 nm whilstthe average diameter of 4-NBAMWCNTs is ca. 60 nm (based on a sample offifty measurements each for native and 4-NBAMWCNTs, standard deviationin both cases was ca. 10 nm).

Numerous research groups have devoted considerable time and effort intomodelling the processes that control CNT formation and growth. A commontheoretical model used to describe CNT formation using chemical vapourdeposition (CVD) as a method of synthesising CNTs is the step-flowgrowth kinetics model. In this model differences in the surfacediffusion rates of carbon atoms along the growing nanotube wall lead tothe formation of regions of multi-island nucleation in front thepropagating “step” which can increase the number of surface defects anddisordered “amorphous” regions. Furthermore, the intrinsic inequality ofsurface diffusional fluxes which feed the growth of different layersduring MWCNT formation lead to “bamboo” structures. In these structuresthe graphite sheets are aligned at an angle to the axis of the nanotubeand thus terminate at the surface of the tube as an edge-plane defect.As nearly every sheet must terminate at the surface, the number ofedge-plane defects is large. One image used to describe these structuresis to liken the bamboo MWCNT to a number of paper cups stacked oneinside the other like so: <<<<<<< where “<” represents multiple nanotubewalls.

The formation of “bamboo-MWCNTs” is temperature dependant. The MWCNTsused in this example were supplied by NanoLab Inc. They manufacturedthis sample using a CVD technique which operated at temperatures of ca.900 K. Theoretical models predict that some multi-island nucleation andthe subsequent formation of bamboo-like regions along the MWCNTs islikely to occur in this temperature region.

It is postulated that it is the presence of these bamboo-like regionsalong the MWCNTs that is responsible for the large uptake of 4-NBA andthe resulting large currents passed during the voltammetric experimentsconducted on them.

In order to provide us with a further insight into the morphology of theMWCNTs, HRTEM imaging was carried out. FIGS. 23A and 23B show the HRTEMimages of a sample of MWCNTs. These images indicate that both“bamboo-like” structures (see below) and hollow-tube structures occupythe length of a MWCNT.

The discovery of “bamboo-like” regions along the MWCNTs provides apossible explanation for the relatively large (milli-Ampere) currentspassed during reduction/oxidation of 4-NBAcarbon and especially4-NBAMWCNTs. These large currents imply that the coverage of 4-NBA onthe graphite particles and MWCNTs is high. If 4-NBA is indeed partiallyintercalating into edge-plane defects on the MWCNTs then these defectsites must be so numerous that they can not simply be located at theopen ends of the nanotubes, but along their surfaces too.

Next X-ray powder diffraction (XPD) was used to examine the effect ofmodifying graphite powder and MWCNTs with 4-NBA. The fundamentalequation used to analyse X-ray diffraction data is given by Bragg's law,equation (2):

nλ=2nd sinθ  (2)

where n is an integer representing the order of reflection from a set ofplanes, L is the wavelength of X-ray radiation/Å, d is the inter-planespacing/Å, and θ is the angle of incidence at which the X-ray radiationfalls on the sample. Any increase in the inter-layer spacing due tointercalation of 4-NBA into the lattice increases the value of d andthus can be observed by comparing the diffractogram of the modifiedsample with the diffractogram of the unmodified sample.

The X-ray powder diffractograms of 4-NBA carbon and unmodified graphitepowder were recorded. Comparison of the diffractograms showed noqualitative or quantitative differences, either in the position of thepeak or in the peak width and shape. Both samples produced a value forthe inter-layer spacing of 3.37 ±0.01 Å which is in good agreement withliterature values of 3.35±0.05 Å. This result excludes the possibilityof full intercalation of 4-NBA into the graphite lattice. However thisdoes not exclude the possibility of partial intercalation of 4-NBA atthe disordered sites containing edge plane steps and defects. This isbecause XPD can only be used to interrogate highly ordered regionspossessing a well defined crystal structure from which to obtainreflected X-rays at well defined values of θ. Any disordered regionssimply scatter the X-rays at random θ values without constructivelyinterfering and hence these signals are lost as noise. Due to the muchmore ordered morphology of MWCNTs compared to graphite powder particlesa comparison of 4-NBAMWCNTs and native MWCNTs using XPD is much moreinstructive as to the nature of the surface modification by 4-NBA.

To this purpose, X-ray powder diffractograms of 4-NBAMWCNTs and “native”unmodified MWCNTs were recorded for comparison (FIG. 24). The firstpoint to mention is that the peak position for both the modified andunmodified MWCNTs is identical within experimental error and correspondsto an inter-layer separation of 3.47 ±0.04 Å which is again consistentwith the literature value of 3.44 ±0.04 Å. The second point to note isin both cases the peaks are broadened and are unsymmetrical with a“tail” on the left hand edge of each peak. This is effect is due tovariations in the inter-layer spacings leading to a certain degree ofdisorder in the crystal structure of the nanotubes. What is important tonote is that the 4-NBAMWCNT peak is considerably broader than the nativeMWCNT peak. Analysis of the normalised peaks reveals that thefull-height-half-width of the native MWCNT peak is 0.9446, whilst the4-NBAMWCNTs full-height-half-width value has increased by nearly fortypercent to 1.3855. This shows that there has been considerabledisruption and an increase in the disorder of the MWCNT structure whichmay provide supporting evidence for partial intercalation of 4-NBAmolecules at the edge-plane surface defects. The increase in disorderingbetween the graphite sheets can be measured quantitatively using theScherrer equation (3) which relates the half-height-full-peak-width tothe average number of planes present in the ordered part of the crystalfrom which the X-ray reflections occur:

$\begin{matrix}{{t = \frac{0.9\mspace{14mu} \lambda}{\beta \; \cos \; \theta}}{and}} & (3) \\{t = {md}} & (4)\end{matrix}$

where λ and θ are defined as per equation (2) above, β is thefull-height-half-peak width, and t is the thickness of the crystallineregion. Hence if the interlayer spacing (d) is known then the number oflayers (m, graphite sheets in this case) in the crystalline, orderedregion can be calculated from (4). Table 1 lists the data obtained fromthe XPD of 4-NBAMWCNTs and native MWCNTs for comparison. The value ofm=9 ordered layers of graphite sheets for native MWCNTs is in agreementwith HRTEM studies of the MWCNTs which found an average of 9 concentrictube walls present. This value has decreased on modifying the CNTs with4-NBA to m=6 which is not an unreasonable value as it is likely thatedge plane defects could expose up to the first three graphite layers.

TABLE 1 A comparison of the experimentally determined X-ray diffractiondata for unmodified native MWCNTs and 4-NBA modified CNTs: Full-m-value, the number of Inter-layer height-half- adjacent ordered layersin spacing/Å width/degrees the crystalline regions Native MWCNT 3.470.9446 9 4-NBAMWCNT 3.47 1.3855 6

Detailed Description of the Third Preferred Aspect of the Invention

The solid state electrochemistry of a pure organic solid abrasivelyimmobilised onto the surface of an electrode and which is in contactwith an electrolyte solution must take place at the three phase boundarybetween the electrode surface|organic solid|solution interface.Concomitant ion insertion from the solution phase into the crystal tomaintain charge neutrality upon oxidation/reduction must occur. FIG. 25schematically depicts, with the white shading, this three-phase boundaryat which transfer of electrons can occur.

However, since electron transfer can only occur at this three-phaseboundary, the electroactive surface area of each individual crystal isrestricted to a very small area which is in contact with both theelectrode surface and the solution.

Accordingly, another approach has been adopted which involves abrasivelyimmobilising a physical mixture of the organic solid and graphite powderonto the surface of a bppg electrode, as shown in FIG. 26.

The electroactive surface area of the electrode shown in FIG. 26 isgreater than that shown in FIG. 25. This is because not only can anelectrode surface|organic solid|solution three phase boundary be formed,but where the carbon particles are in contact with the organic solidcrystals an additional carbon particle organic solid I solution threephase boundary is formed, thus allowing increased charge transfer.However, while electroactivity is improved using this method, the degreeof contact between the graphite particles and the organic crystals isrestricted by the size of the graphite particles.

The third preferred aspect of the present invention is concerned withthe use of agglomerates comprising carbon nanotubes dispersed in abinder, wherein the binder is a redox-active material. The carbonnanotubes are preferably multi-walled carbon nanotubes (MWCNTs).

Electrodes made from the agglomerates comprise carbon nanotubes and aredox active material disposed on a substrate. The carbon nanotubes andredox active material need not be mixed in the form of an agglomerate.Instead they may simply be abrasively immobilised on the surface of thesubstrate. However, it is preferred that the carbon nanotubes and redoxactive material are disposed on the electrode in the form of anagglomerate, as described above in the first embodiment of theinvention. Furthermore, it is again preferred that the carbon nanotubesare in the form of MWCNTs.

The electroactivity of these electrodes is greater than prior artelectrodes. As a result, a smaller amount of material is required toachieve the same response and hence the electrodes themselves can bereduced in size, allowing for miniaturisation of the electrochemicalsensors in which they are employed.

A method for preparing an agglomerate for use in electrochemical sensorscomprises dispersing multi-walled carbon nanotubes in a binder, whereinthe binder is a redox active material. This agglomerate can then be usedin a method for preparing an electrode, which method comprises providinga substrate and applying carbon nanotubes and a redox active material tothe surface of the substrate.

The agglomerates and electrodes can be used in electrochemical sensors.Such sensors can be used to monitor a number of different species, suchas pH, carbon monoxide, hydrogen sulphide, oxygen, carbon dioxide, metalions etc. The sensors are particularly useful in the measurement of pH,a measurement that is important in a number of fields, such asenvironmental, chemical, waste water, industrial and effluent.

By preparing electrodes having carbon nanotubes and redox activematerials applied to a substrate, electroactivity is significantlyimproved because greater contact with the surface of the redox activematerial is achieved. In effect, the nanotubes act as “molecular wires”forming numerous three phase boundaries between the nanotube|redoxactive materiallsolution. When an agglomerate of nanotubes and redoxactive material is prepared even greater electroactivity is achieved. Inthis case the nanotubes are cemented together in bundles by the redoxmaterial so that they not only run along the surface, but also into andthrough the agglomerate. At every point of contact between the organicsolid, nanotube and the solution, a three-phase boundary is formed. Thisresults in a much larger electroactive surface area compared to theprior art.

Thus, an advantage of the present invention is that the electroactivearea is increased, meaning that a smaller amount of material is requiredin order to achieve the same effect. As a result, the electrodes, aswell as the sensors in which these electrodes are employed, may bereduced in size. Miniaturisation of sensors increases the number ofapplications for which they can be applied. For example, they could beused in biomedical applications where it may be necessary to introducethem into the patient's body. Alternatively they could be used for otherapplications where sensing apparatus must be used in confined spaces.Miniaturisation also allows for greater portability of the sensors.

The relative proportions of the nanotubes and redox active material usedin the invention can be varied by the person skilled in the art. When amechanical mixture of nanotubes and redox active material is immobilisedon a substrate the ratio of these components is preferably 10:1 to 1:10by mass, more preferably 5:1 to 1:5, more preferably 1:2 to 2:1. In thecase where the components are present on the substrate in the form of anagglomerate, the relative proportions may vary significantly. Inprinciple there is no lower limit to the amount of carbon nanotubespresent.

The individual components and aspects of the third aspect of theinvention will now be described in more detail.

Carbon Nanotubes

Carbon nanotubes (CNTs, also referred to herein as nanotubes) have beenknown for a number of years, having been discovered in 1991 (see S.Iijima, Nature, 1991, 56, 354). One field that has seen a largeexpansion in the study and use of nanotubes is electrochemistry. Carbonnanotubes are particularly useful in this field due to their notedmechanical strength, structure and good electrical conductivity. Theseproperties have been used in electroanalytical applications ranging fromcatalytic detection and analysis of biological molecules such asdopamine, cytochrome c and carbohydrates, to the sensing of analytessuch as hydrogen peroxide, hydrazine and TNT.

Structurally, nanotubes approximate to “rolled up” sheets of graphiteand as such are relatively hydrophobic in nature. There are two mainconfigurations of these “rolled up” sheets: single-walled carbonnanotubes and multi-walled carbon nanotubes (MWCNTs). In the presentinvention either configuration may be used.

Suitable nanotubes include those purchased from Nanolab Inc. (Brighton,MA, USA). The physical properties of the nanotubes can be optimised bythe person skilled in the art, although exemplary nanotubes have adiameter of from 1 to 50 nm, preferably from 5 to 30 nm, and a length offrom 1 to 50 nm, preferably from 5 to 30 nm. Preferably the carbonnanotubes have a relatively high purity, preferably from 80 to 100%,more preferably from 90 to 100%, most preferably from 95 to 100%.

Redox Active Material

The redox active material may be any organic material capable ofundergoing electron loss and gain. Preferably the redox active materialis a solid phase material. When immobilised onto a substrate, e.g.glassy carbon or a basal plane pyrolytic graphite (bppg) electrode, theyundergo concomitant proton and electron loss/gain onoxidation/reduction.

In order to be used in electrochemical sensors, at least a part of theredox active material will need to be sensitive to the species which isto be detected or measured. It is preferred that the electrodes beuseful in the manufacture of pH meters, and accordingly it is preferredthat the peak potential of the redox active materials depends on thelocal proton concentration.

The voltammetry of such compounds sensitive to pH, when immobilised asmolecular solids onto the surface of an electrode, has been found toexhibit Nernstian behaviour which can be described according to thefollowing Nernst equation:

$E_{p} = {E_{f}^{0} - {\frac{2.3\mspace{14mu} {RTm}}{nF}{pH}}}$

where E_(p)/V is the peak potential, E_(f) ⁰/V is the formal potentialof the redox couple, R/J K⁻¹ is the universal gas constant, T/K is thetemperature and m and n are the number of protons and electrons involvedin the redox process respectively. In the materials tested in theExamples which follow, m and n are often equal to 2.

Accordingly, by studying the voltammetric response of these compounds,for example using cyclic voltammetry or square-wave voltammetry, alinear response of peak potential to pH would be expected.

The redox active material can comprise more than one compound. Forexample, the material may comprise a chemically sensitive redox materialand a chemically insensitive redox material. In this embodiment, thechemically insensitive redox material serves as a reference material. Bymeasuring the potential difference between the current peaks for thechemically sensitive and chemically insensitive redox materials, theconcentration of the species to be measured can be determined.

Alternatively, the redox active material may comprise more than onechemically sensitive material which is sensitive to the same species. Bymeasuring the potential difference between the current peaks for thechemically sensitive materials compared to that of the referenceelectrode, a more accurate concentration of the species to be measuredcan be obtained.

The redox active materials used in the example are preferablyhydrophobic, have a low solubility in water. This allows them, when anagglomerate is being manufactured, to mix with the carbon nanotubes insolution and results in the agglomerate precipitating out of solutionwhen an excess of aqueous solution is added.

Suitable redox active materials include quinones and anthracenes, forexample 9,10-anthracene, 9-nitroanthracene, phenanthraquinone (PAQ) and1,2-napthaquinone (NQ). Other materials that can be used includeazobenzene, diphenylamine, methylene blue, 3-nitrofluoranthene,6-nitrochrysene and thionin.

The Agglomerate

The agglomerate of the invention comprises nanotubes and a binder,wherein the binder is a redox active material. The nature of thenanotubes and redox active material may be as described above.

The agglomerate is made by dispersing the nanotubes in a binder. Thepreferred method comprises combining the nanotubes and binder materialin a solvent, and then precipitating the agglomerate out of thesolution. In particular, the method may comprise:

-   -   (1) combining the carbon nanotubes and the binder in a solvent;    -   (2) adding an excess of aqueous solution in order to cause        precipitation of the agglomerate out of the solvent; and    -   (3) recovering the agglomerate.

Preferably the solvent is a hydrophobic solvent, comprising smallorganic molecules. The solvent should be chosen such that the redoxactive compound and the carbon nanotubes are both soluble within it.Suitable solvents include all common organic solvents such as acetone,acetonitrile and dimethyl formamide.

The agglomerate preferably comprises the carbon nanotubes and redoxactive material only, with no other materials present. However, theagglomerate may contain some impurities such as residual solvent, leftas a result of a process by which the agglomerate is be produced.Preferably these impurities comprise less than 1 wt % of theagglomerate, more preferably less then 0.5 wt %. The precise level ofimpurities which is acceptable in the agglomerate will depend upon howthe impurities affect the voltammetry of the agglomerate.

The size of the agglomerates depends upon the nature and proportions ofthe components used in their preparation and the conditions of theprocess by which they are prepared. However, exemplary agglomerates maybe approximately 10 μm in diameter and consist of bundles of nanotubesrunning into and throughout an amorphous molecular solid which binds theagglomerate together.

The Substrate

The substrate onto which are applied the carbon nanotubes and redoxactive material may be any substrate conventionally used in themanufacture of electrodes. For example, the substrate may be a basalplane pyrolytic graphite (bppg) electrode or glassy carbon, metalelectrodes such as gold or platinum, or optically transparent electrodessuch as those comprising ITO. The substrate preferably has goodelectrical contact with the carbon nanotubes, and also has a surfacesuch that good coverage with the carbon nanotubes and redox activematerial can be achieved.

The Sensor

The structure of the sensor will depend upon its final application, andhence depends upon the substance which it is to measure and theenvironment in which measurement will take place. Known sensorstructures may be employed in conjunction with the agglomerates andelectrodes described herein.

Exemplary sensors may have a two or three terminal arrangement. Thus,they may comprise a working electrode of the invention and a combinedcounter and reference electrode, or a working electrode, counterelectrode and a reference electrode. The reference electrode and counterelectrode can be any conventional electrodes known in the art.

The materials used in the sensor depend upon which species the sensor isintended to measure and the environment in which the sensor is to beused. In order to modify the sensor to be sensitive to a differentspecies it is simply required for the skilled person to substitute theredox active material with a different redox active material sensitiveto the species which is to be measured.

Examples of the Third Preferred Aspect of the Invention Example 9Formation of Nanotube Agglomerates and their Abrasive Immobilisationonto the Surface of Basal Plane Pyrolvtic Graphite

Reagents and Equipment

All reagents were obtained from Aldrich (Gillingham, UK) with theexception of potassium chloride which was obtained from Riedel de Haën(Seelze, Germany) and were of the highest grade available and usedwithout further purification. All aqueous solutions were prepared usingdeionised water from an Elgastat (Elga, UK) UHQ grade water system witha resistivity of not less than 18.2 ME cm. All cyclic voltammetricmeasurements were made after degassing the solution with pure N₂ gas(BOC Gases, Guildford, Surrey, UK) for 30 minutes and unless otherwisestated were recorded at a temperature of 20±2° C.

Multi-walled carbon nanotubes (MWCNTs) with purity ˜95% were purchasedfrom Nanolab Inc. (Brighton, Mass., USA) and were used without furtherpurification.

Solutions of known pH in the range pH 1-12 were made up in de-ionisedwater as follows: pH 1, 0.1M HCl; pH 4.6. 0.1M acetic acid+0.1M sodiumacetate; pH 6.8, 0.025 M Na₂HPO₄+0.025 M KH₂PO₄; pH 9.2, 0.05 M disodiumtetraborate; pH 12 0.01M sodium hydroxide. These solutions contained inaddition 0.1M KCl as additional supporting electrolyte. pH measurementswere performed using a Jenway 3030 pH meter.

Electrochemical measurements were recorded using a μ,Autolab computercontrolled potentiostat (Ecochemie, Netherlands) with a standardthree-electrode configuration. All experiments were carried out using adouble-walled glass cell of volume 25 cm³ thermostatted to the desiredtemperature (20-70° C.) through circulation of water from a heated bath.A basal plane pyrolytic graphite electrode (bppg, 0.20 cm², Le CarboneLtd., Sussex, UK) acted as the working electrode (see below). A platinumcoil acted as the counter electrode and a saturated calomel electrode asthe reference electrode (SCE, Radiometer, Copenhagen) completed the cellassembly.

Unless stated otherwise cyclic voltammograms were recorded using thefollowing parameters: step potential 2 mV, scan rate 50 mV s⁻¹. Squarewave voltammetric parameters were as follows: frequency 12.5 Hz, steppotential 2 mV and amplitude 25 mV. Scanning electron microscopy (SEM)was conducted using a Jeol 6500F instrument.

It is worth noting that the toxicology of both 9,10-phenanthraquinoneand 1,2 napthaquinone has not yet been fully investigated. Bothcompounds may be harmful, or irritant by skin contact or inhalation andare suspected carcinogens.

Formation of Nanotube Agglomerates and their Abrasive Immobilisationonto the Surface of basal plane pyrolytic graphite

Agglomeration of MWCNTs with either 9,10-phenanthraquinone (PAQ) or1,2-napthaquinone (NQ) was achieved by mixing 50 mg MWCNTs with 10 cm³of a 10 mM solution of either PAQ or NQ in acetone and slowly adding 25cm³ of 0.1M HCl+0.1M KCl aqueous solution. The reaction mixture wasstirred continuously for 2 hours in a beaker and then filtered bysuction after which it was washed with distilled water to remove theacid and salt. It was then air-dried by placing inside a fume hood for12 hours and finally stored in an airtight container until required.

The agglomerates of nanotubes and either PAQ or NQ were then abrasivelyimmobilised onto the surface of the bppg electrode prior tocharacterisation. This was done by initially polishing the electrode onglass polishing paper (H00/240), after which it was polished on siliconcarbide paper (P1000C) for smoothness. The nanotube-PAQ or nanotube-NQagglomerates were then mechanically immobilised onto the bppg electrodeby gently rubbing the electrode surface on a fine filter paper (Whatman)containing the agglomerates.

Example 10 Characterisation of the Nanotube Agglomerates

First the formation of the agglomerates was verified using SEM and CVand then the immobilisation of the agglomerates onto the surface of thebppg was confirmed voltammetrically using CV.

Determination of the Degree of Agglomeration

In order to verify that agglomerates of PAQ or NQ with MWCNTs are formedrather than a physical mixture of microcrystals of either PAQ or NQ andMWCNTs, scanning electron microscopy (SEM) was employed. Each materialwas imaged separately after abrasive immobilisation onto the surface ofa clean bppg electrode had been carried out. First the SEM image of pureMWCNTs (with no organic solid present) was recorded (FIG. 29). Next theSEM images of the MWCNTs, which had been modified according to theprocedure given in section 2.2 were recorded for comparison (FIGS. 30and 31).

These images confirm that unlike unmodified MWCNTs on bppg which aredispersed evenly over the surface (FIG. 29), the MWCNT-PAQ material hadindeed formed agglomerates of nanotubes cemented together in bundles bythe PAQ (FIGS. 30 and 31) as shown schematically in FIG. 28.

Finally, the cyclic voltammograms (overlaid in FIG. 35) of PAQ crystals,a mechanical mixture of MWCNTs and PAQ crystals, and the MWCNT-PAQagglomerates were recorded separately in pH 6.8 buffer after abrasiveimmobilisation onto the surface of a bppg electrode.

In each case a nearly symmetrical wave shape with similar peak heightswas observed at ca −0.23 V vs. SCE, with a slight peak to peakseparation of ca. 20 mV.

Experimentally it is impossible to control the exact amount of materialthat is abrasively immobilised onto the surface of the bppg electrode.However, as is apparent from FIGS. 8, 9 and 10, visibly less of theMWCNT-PAQ agglomerate material was needed on the surface of theelectrode to give similar magnitudes of peak currents as either the PAQcrystals on their own. FIG. 30 shows that the actual surface coverage ofthe bppg electrode by the MWCNT agglomerates is sparse. The mechanicalmixture of MWCNTs and PAW crystals gives an intermediate result.

A crude calculation of the effective electroactive surface area of theMWCNT-PAQ agglomerate was undertaken. The PAQ molecules were consideredto occupy rectangles of area 2.5×10⁻¹⁹ m² and the number of moleculesoxidised or reduced was calculated from the peak area of thecorresponding cyclic voltammogram. Thus the approximate electroactivesurface area of this material was found to be typically greater thanthree times that of the bppg electrode itself.

It is worth noting that this crude calculation assumes the area of theelectrode surface to be equal to its geometric area. In reality,polished surfaces may have a surface area substantially greater than thegeometric area. Furthermore a proportion of the PAQ or NQ binder may notreact due to inefficient charge transfer to the electrode surface, asimpurities on the MWCNT surface may impair conductivity at thebppg/MWCNT interfacial region. However, even with these limitations inmind the results are still indicative of a significant increase in theelectroactive surface area of the electrode when MWCNT agglomerates areimmobilised upon it.

One possible explanation for this is found by considering that one ofthe advantages of electrodes modified with carbon nanotubes is a largereffective electroactive surface area with obvious analytical benefits.The structure of abrasively immobilised MWCNT agglomerates has beenshown (above) to consist of bundles of MWCNTs cemented together in anamorphous organic solid on the surface of a substrate electrode. Asdiscussed earlier, such a structure confers a significantly greaterelectroactive surface area where three-phase boundaries can be formedthan the situation when pure organic crystals or physical mixtures of anorganic solid and graphite powder are used. Hence less of theagglomerate material is required to produce signals of similar orders ofmagnitude as in the other two cases studied above.

Determination of Surface Immobilisation

The immobilisation onto the bppg surface was confirmed using cyclicvoltammetry. The two agglomerates were then studied over the entire pHrange pH 1-12. The first step in the protocol, once the potential regionof the redox process for PAQ or NQ had been determined, was to conducttwenty repetitive scans (the exact potential range of these scans variedbetween PAQ and NQ and with pH) to ensure the stability of the species.In the case of both MWCNT-PAQ agglomerates and MWCNT-NQ agglomerates anearly symmetrical wave shape with a slight peak separation thatincreased with increasing scan rate (see below) was observed at every pH(FIGS. 36A and 36B). It was found that after twenty repetitive scans thepeak currents (which initially were found to decrease slightly) remainedstable and that the charges (peak areas) of both the oxidative andreductive peak processes were equal to each other.

The next step in the protocol was to replace the electrolyte solutionwith fresh solution and record the voltammetric response. Thecorresponding cyclic voltammogram (overlaid in FIGS. 36A and 36B) wasfound to overlay the last scan thereby confirming that the electroactivespecies remains on the surface of the electrode and is not released tosolution.

Finally the scan rate was varied from 25 to 900 mV s⁻¹ (FIGS. 37A and37C) and a plot of peak current vs. scan rate was found to be almostlinear (FIGS. 37B and 37D). The peak separation (ca. 20 mV at low scanrate) is close to the ideal zero peak to peak separation for animmobilised, electrochemically reversible species. However, thediscrepancy between the experimental and theoretical peak separation,and the deviation from linearity in the plots of peak current vs. scanrate may be tentatively attributed to some slight ohmic distortionand/or electrode kinetic factors. In fact the wave shapes and thevariation of peak potential with increasing scan rate suggest that anelectrochemically quasi-reversible system exists over the entire pHrange studied.

Example 1 Voltammetric Response of Agglomerates to pH at RoomTemperature

Having established, using cyclic voltammetry in Example 2 above, that astable, electrochemically almost reversible system is observed for bothMWCNT-PAQ and MWCNT-NQ agglomerates over the entire pH range from pH 1to pH12, square wave voltammetry was utilised as the electrochemicalmethod of probing the system in all the studies detailed below. This hassignificant advantages as compared to conventional cyclic voltammetry,since it provides a means of carrying out a single sweep which producesa well-defined voltammetric peak due to PAQ and NQ having almostreversible electrode kinetic behaviour. This can therefore aid in theresolution of the MWCNT-PAQ and MWCNT-NQ reduction waves, especially athigher pH where oxygen reduction may compete at a similar potential tothat of the redox process of interest. Square wave voltammograms wererecorded for MWCNT-PAQ and MWCNT-NQ agglomerates in a range of pHsolutions (pH 1, 0.1M HCl; pH 4.6, 0.1M acetic acid+0.1M sodium acetate;pH 6.8, 0.025 M Na₂HPO₄+0.025 M KH₂PO₄; pH 9.2, 0.05 M disodiumtetraborate; pH 12 0.01M sodium hydroxide) and are overlaid in FIGS. 38Aand 38B respectively. It is apparent from FIGS. 38A and 38B that as thepH is increased the peak potential of MWCNT-PAQ and MWCNT-NQ shifts tomore negative potentials as expected. This behaviour is consistent withthat observed for PAQ crystals abrasively immobilised on a bppgelectrode.

The corresponding plot of peak potential against pH reveals a linearresponse from pH 1 to pH12 with a gradient of 55.2 and 53.2 mV/pH unitfor MWCNT-PAQ and MWCNT-NQ respectively, which is reproducible uponrepetitive electrode preparations as described earlier.

This is close to a Nernstian response as given by the equation discussedearlier and is again consistent with previous studies on carbon powder.

Example 12 Voltammetric Response of Agglomerates to pH at ElevatedTemperature

The effect of temperature on the voltammetric response of theagglomerates was investigated in order to determine whether thematerials might be used as suitable pH probes for high-temperatureenvironments.

One factor to be noted while studying the effect of temperature on thesystem is how the pH of the solution varies with temperature as thedissociation constants of the components in the buffer solution varywith temperature. To this end three IUPAC buffers (pH 4.6, pH 6.8 and pH9.2) were utilised that have a known set of pHs at a given temperature.The error due to using pH1 and pH12 solutions of dilute HCl or NaOHrespectively for high-temperature studies is negligible as the pHvariation with temperature of all buffers is small and these are theextremities of the plot.

Square wave voltammograms were recorded for each pH studied in the rangepH I to pH 12 over the temperature range 20-70° C. for both MWCNT-PAQand MWCNT-NQ. Note that the upper limit of this temperature range waslimited by the onset of bubble formation on the electrode as thetemperature approached the boiling point of the solution. FIGS. 15A and15B show the overlaid square wave voltammograms of MWCNT-PAQ andMWCNT-NQ respectively over the temperature range 20-70° C. in pH 6.8IUPAC buffer. Similar responses were obtained at every other pH studied.There is a shift of the peak potential to more negative values withincreasing temperature which can be attributed in part to a combinationof changes in the SCE reference couple, the temperature dependence ofthe formal potential (E_(f) ⁰) and the temperature term in the Nernstianequation discussed earlier. It is worth noting that the peak currentinitially increases with temperature, but then decreases steadily after30° C. In order to investigate the stability of the agglomerates atelevated temperatures cyclic voltammetry was employed at everytemperature studied in the range 20-70° C. Five hundred scans wereperformed at each temperature on the abrasively immobilised MWCNTagglomerates at a scan rate of 200 mV s⁻¹ and every fifth scan recorded,corresponding to a time interval of ca. 35 seconds. In every case asingle, reversible and almost symmetrical wave was observedcorresponding to the MWCNT-PAQ or MWCNT-NQ agglomerate as describedearlier. From these voltammograms the peak area was measured, which isproportional to the amount of the electroactive species remaining on thesurface of the electrode in the form of MWCNT agglomerates. From a plotof peak area against time (not shown) it was apparent that although thesignal remains stable for 500 scans over ca. 1 hour at 20° C., attemperatures above 30° C. there is a decrease in the magnitude of thesignal. This behaviour is in contrast to chemically adsorbedanthraquinone on graphite particles, where the magnitude of the signalincreases steadily with increasing temperature, but is in agreement withearlier studies of PAQ physically adsorbed onto the surface of graphiteparticles. This would suggest that the decrease in signal observed atelevated temperatures for MWCNT-PAQ and MWCNT-NQ agglomerates is due topartial dissolution of the agglomerates. However, even at 70° C. after500 scans (ca. 1 hour) an appreciable signal, ca. 10% of the originalvalue, still remains.

FIG. 40 shows the overlaid square wave voltammogram for NQ at 70° C.showing that an analytically useful response may be obtained at thiselevated temperature.

A plot of peak potential vs. pH at each temperature studied yielded astraight line, with R² values not less than 0.998, for both MWCNT-PAQand MWCNT-NQ agglomerates, the gradients of which are given in Table 6.The theoretical gradient as predicted by the Nernst equation is alsogiven in Table 6 for comparison. As can be seen the variation of thegradient of peak potential with pH is not Nernstian and indeed isrelatively insensitive of temperature varying by ca 3 mV/pH unit over atemperature range of 50° C. This is advantageous in that it not onlydemonstrates that these agglomerates may be used as pH sensors atelevated temperatures, but also that they are not greatly affected byquite significant changes in temperature.

TABLE 6 Experimental gradients of plots of peak potential against pH ateach temperature studied for abrasively immobilised MWCNT-PAQ andMWCNT-NQ agglomerates. Theoretical Experimental Gradient Temperaturegradient (mV/pH unit) (K) (mV/pH unit) MWCNT-PAQ MWCNT-NQ 293 58.1 55.253.2 303 60.1 55.4 53.4 313 62.1 56.5 53.7 323 64.1 56.7 53.9 333 66.156.9 54.9 343 68.1 57.0 56.6

As shown in Examples 11 and 12, a linear response of peak potential topH is observed for both MWCNT-PAQ and MWCNT-NQ agglomerates over theentire pH range and temperature range studied. Furthermore, a comparisonof the gradients of a plot of peak potential against pH at eachtemperature reveals that both MWCNT-PAQ and MWCNT-NQ agglomerates arerelatively insensitive to the effects of temperature changes. Such aproperty is advantageous for pH measurement where samples are obtainedat a range of different temperatures.

Detailed Description of the Fourth Preferred Aspect of the Invention

According to a fourth preferred aspect of the invention, the electrodecomprises a layer on a substrate of a composition of the carbon and theredox-active compound, the layer having an edge formed by cuttingthrough the layer to expose carbon and redox-active compound.

An electrode of the fourth preferred aspect of the invention can be madeby first printing onto a substrate using a carbon based inked mixed withcrystals if whichever chemical is to be used for the analysis. Theprinted electrode is then laminated and shaped into a strip, as shown inFIG. 41. The electrode is activated by cutting the end off the strip,with a scissors, to reveal a cross section of the print. This crosssection will then have a carbon (ink) and crystals of the chemical thatwas mixed with the ink, exposed at its surface, as shown in FIG. 42.

When the cut electrode is then dipped into solution, reactions can beinduced to happen at the triple phase boundary between the carbon, thesolid crystals and the analyte solution.

The increased sensitivity associated with the “cut” electrode can beseen in FIGS. 43 and 44.

FIG. 43 shows a Square Wave Voltammagram (SWV) recorded using a printedelectrode (lower line). The figure also shows that the SWV recorded withthe same electrode after the end had been cut off (upper line). Thesharp peak visible on the SWV for the cut electrode (upper line) is dueto the exposed crystals in contact with the solution.

When the printed electrodes have been laminated there is no surface ofthe electrode exposed. However when the end is cut off the electrodestrip, the exposed surface (see FIG. 41) produces a SWV like the onepresented in FIG. 44, the lowest line. Also shown in FIG. 44 are thecurves from FIG. 43 for comparison. The plot also demonstrates thecleaner signal achieved with the laminated and cut electrode.

It will be apparent to those skilled in the art that modifications maybe made to the invention as described above without departing from thescope of the claims below.

1. A method for preparing an electrode for use in an electrochemicalsensor, said method comprising modifying carbon with a chemicallysensitive redox active material wherein the material is a single redoxactive species or two redox active species, with the proviso that thematerial that is two redox active species does not includeanthraquinone, phenanthrenequinone or N,N═-diphenyl-p-phenylenediamine(DPPD), and wherein the electrode is operable over an entire pH range of1 to
 12. 2. A method according to claim 1, wherein the step of modifyingthe carbon comprises one or more of the following methods: 1)derivatisation via physical adsorption of the chemically sensitive redoxactive material; and 2) physical mixing with the chemically sensitiveredox active material and a binder.
 3. The method according to claim 1,further comprising the step of applying the carbon modified with thechemically sensitive redox active material to a substrate.
 4. The methodaccording to claim 3, wherein the step of applying comprises abrasivelyimmobilising the composition on the surface of the substrate.
 5. Amethod for preparing an electrode for use in an electrochemical sensoras claimed in claim 2, said method comprising modifying carbon with asingle chemically sensitive redox active material selected from ananthraquinone, phenanthrenequinone or N,N-diphenyl-p-phenylenediamine(DPPD).
 6. An electrode for use in an electrochemical sensor, saidelectrode comprising carbon and a redox-active compound wherein theredox-active compound is a single redox active compound or two redoxactive compounds with the proviso that if two redox active compounds areprsent, then the compounds are not selected from anthraquinone,phenanthrenequinone or N,N′-diphenyl-p-phenylenediamine (DPPD), andwherein the electrode is operable over an entire pH range of 1 to
 12. 7.The electrode according to claim 6, wherein the redox active compound isnot anthraquinone, phenanthrenequinone orN,N′-diphenyl-p-phenylenediamine.
 8. The electrode according to claim 6,wherein the redox active compound is a chemically sensitive compound. 9.The electrode according to claim 6, comprising carbon modified with aredox active material, wherein the redox active material is a chemicallysensitive material which undergoes an irreversible chemical reactionwhen the electrode is subjected to cyclic voltammetry.
 10. The electrodeaccording to claim 9, wherein the product of the irreversible chemicalreaction displays reversible electrochemistry when the electrode issubjected to cyclic voltammetry.
 11. The electrode according to claim 6,wherein the chemically sensitive redox active compound is sensitive tothe concentration of protons.
 12. The electrode according to claim 9,wherein the chemically sensitive redox active material undergoespolymerization when subjected to cyclic voltammetry.
 13. The electrodeaccording to claim 12, wherein the chemically sensitive redox activematerial has a nitro group substituent.
 14. The electrode according toclaim 6, wherein the electrode comprises a single redox active compoundselected from the group consisting of anthraquinone, phenanthrenequinoneor N,N′-diphenyl-p-phenylenediamine.
 15. The electrode according toclaim 6, wherein the chemically sensitive redox active material isphysically adsorbed to the carbon.
 16. An electrochemical sensorcomprising an electrode as claimed in claim
 6. 17. An electrode for usein a electrochemical sensor, said electrode comprising carbon and asingle redox-active compound selected from anthraquinone,phenanthrenequinone or N,N′-diphenyl-p-phenylenediamine (DPPD); whereinthe chemically sensitive redox active material is physically adsorbed tothe carbon; and wherein the electrode is operable over an entire pHrange of 1 to
 12. 18. A method for preparing an electrode for use in anelectrochemical sensor, said method comprising modifying carbon with asingle chemically sensitive redox active material selected from thegroup consisting of anthraquinone, phenanthrenequinone orN,N-diphenyl-p-phenylenediamine (DPPD); and wherein the step ofmodifying the carbon comprises one or more of the following methods: 1)derivatisation via physical adsorption of the chemically sensitive redoxactive material; and 2) physical mixing with the chemically sensitiveredox active material and a binder; and wherein the electrode isoperable over an entire pH range of 1 to
 12. 19. A method according toclaim 17, wherein the step of modifying the carbon comprisesderivatisation via physical adsorption of the chemically sensitive redoxactive material.
 20. An electrochemical sensor comprising an electrodeas claimed in claim 17.